RU2377303C2 - Wheat with modified activity of brahching enzyme, and also starch and starch-containing products produced from it - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: genetic modification of wheat in the form of mutation in gene SBEIIa with reduction of level of its activity.

EFFECT: grain with high content of amylose in its starch; wheat with reduced level of SBEIIb-activity with grain with non-wrinkled phenotype, with relatively high content of amylose.

62 cl, 28 dwg, 12 tbl, 16 ex

Description

2420-135359RU / 092

WHEAT WITH MODIFIED ACTIVITY OF BRANCHING ENZYME,

AND ALSO RECEIVED FROM IT STARCH AND STARCH-CONTAINING PRODUCTS

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a wheat plant, the grain of which contains starch with a high content of amylose. The present invention also relates to wheat with reduced activity of starch-branching enzyme IIa (SBEIIa) in the endosperm and to methods for producing such plants. In addition, the present invention relates to grain, as well as to starch and to food and non-food products derived from this grain.

State of the art

The starch content in the grain is approximately 45-65% of the total mass of mature grain. Starch consists of two types of molecules, amylose and amylopectin. Amylose is basically a linear molecule consisting of glucoside chains linked by an α-1,4 bond, and amylopectin is a highly branched molecule having linear chains linked by α-1,6 glucosidic bonds.

The synthesis of starch in the endosperm of higher plants is carried out by a number of enzymes that catalyze four key stages. In the first step, ADP-glucose pyrophosphorylase activates the monomeric starch precursor by synthesizing ADP-glucose from G-1-P and ATP. In the second stage, the activated glucose donor, ADP-glucose, is transferred by starch synthases to the non-reducing end of the already existing α1-4 bond. In a third step, starch-branching enzymes introduce branch points by cleaving the region of α-1,4-linked glucan, followed by transferring the cleaved chain to an acceptor chain to form a new α-1,6-bond. Starch-branching enzymes are only those enzymes that can introduce α-1,6 bonds into α-polyglucans, and therefore they play an important role in the formation of amylopectin. Finally, starch-branching enzymes remove some of these bonds at the branch point, although the mechanism of such removal is still unclear (Myers et al., 2000).

Although it is obvious that for the normal synthesis of starch granules in higher plants, at least these four activities are necessary, however, many isoforms of each of these four activities were found in the endosperm of higher plants, and based on the analysis of mutations (Wang et al., 1998 , Buleon et al., 1998) or from an analysis carried out by modifying gene expression levels using transgenic technology (Abel et al., 1996, Jobling et al., 1999, Scwall et al., 2000), it has been suggested that each individual isoform plays a role in this synthesis. However, the specific role of each isoform in the activity of each enzyme involved in starch biosynthesis is still unknown, and it is also unknown whether these roles have noticeable differences for different species. In the cereal endosperm, there are two isoforms of ADP-glucose pyrophosphorylase, one of which is in the amyloplast and the other in the cytoplasm (Denyer et al., 1996, Thorbjornsen et al., 1996). Each form consists of two types of subunits. Mutants leading to wrinkling, ( sh2), and brittleness, (bt2) , corn grains, are damaged in the large and small subunits, respectively (Giroux & Hannah, 1994). Four classes of starch synthase were found in the endosperm of the grain: an isoform that is localized exclusively in the starch granule, namely granule-associated starch synthase (GBSS); two forms that are distributed between the starch granule and the soluble fraction (SSI, Li et al., 1999a, SSII, Li et al., 1999b), and the fourth form, which is completely localized in the soluble fraction of SSIII (Cao et al., 2000, Li et al., 1999b, Li et al., 2000). GBSS has been shown to play a major role in the synthesis of amylose (Shure et al., 1983), and it has been shown that mutations in SSII and SSIII lead to a change in the structure of amylopectin (Gao et al., 1998, Craig et al., 1998) . In this case, no mutations were described that would play a decisive role in SSIII activity.

Three forms of a branching enzyme are expressed in the cereal endosperm, namely, branching enzyme I (SBEI), branching enzyme IIa (SBEIIa) and branching enzyme IIb (SBEIIb) (Hedman & Boyer, 1982, Boyer & Preiss, 1978, Mizuno et al., 1992 , Sun et al., 1997). Genomic sequences and cDNA sequences have been characterized for rice (Nakamura & Yamanouchi, 1992), maize (Baba et al., 1991, Fisher et al., 1993; Gao et al., 1997) and wheat (Repellin et al., 1997 ; Nair et al., 1997; Rahman et al., 1997). Comparison of these sequences by alignment showed a high degree of similarity at both the nucleotide and amino acid levels, and allowed us to group these enzymes into classes SBEI, SBEIIa and SBEIIb. The sequences SBEIIa and SBEIIb show approximately 80% identity with each other, and in particular in the central regions of the genes. SBEIIa and SBEIIb may also differ in the type of expression. In general terms, SBEIIb is specifically expressed in the endosperm, while SBEIIa is present in all plant tissues.

In wheat endosperm SBEI (Morell et al., 1997) is found exclusively in the soluble fraction, while SBEIIa and SBEIIb are found both in the soluble fraction and in the fraction associated with starch granules (Rahman et al., 1995). In maize and rice, the production of high amylose phenotypes has been shown to be caused by damage to the SBEIIb gene, also known as the amylose extension gene (ae), (Boyer & Preiss, 1981, Mizuno et al., 1993; Nishi et al. , 2001). In these SBEIIb gene mutants, starch granules in the endosperm exhibit an abnormal morphology with significantly increased amylose content, and the level of branching of residual amylopectin was lower and the number of short chains was reduced (<DP17, and especially DP8-12). In addition, the gelatinization temperature of starch increased. Moreover, there is a significant pool of material that has been defined as an “intermediate”, that is, an intermediate between amylose and amylopectin (Boyer et al., 1980, Takeda et al., 1993b). In contrast, maize plants having a mutation in the SBEIIa gene caused by the insertion element of the mutator gene (Mu) and therefore not expressing SBEIIa protein did not differ from wild-type plants in terms of starch branching in the endosperm (Blauth et al., 2001), although they differed in starch in the leaves. Similarly, in rice plants deficient in SBEIIa activity, there was no significant change in the profile of the amylopectin chain in the endosperm (Nakamura 2002). In these maize and rice plants, the SBEIIa and SBEIIb genes were not linked in the genome.

In maize, the dull1 mutation leads to a decrease in starch content and to an increase in amylose levels in the endosperm, where the degree of change depends on the genetic background, as well as an increase in the level of branching in residual amylopectin (Shannon & Garwood, 1984). The gene corresponding to the mutation was identified and isolated using the transposon tagging strategy with a transposon mutator (Mu), and it was shown that it encodes an enzyme called starch synthase II (SSII) (Gao et al., 1998). In cereals, this enzyme is currently known as a member of the SSIII family (Li et al., 2003). The mutant endosperm had reduced levels of SBEIIa activity associated with the dull1 mutation. In other crops, no similar mutation has been reported. It is not known whether these data relate to other crops, such as wheat.

WO 94/09114 suggested the use of sense and antisense genes to alter the natural ratios of starch synthase (SS) and SBE in corn. However, there was no evidence of the proposed molecular strategies, and no specific decrease in SBEIIa activity was suggested.

In potatoes, feedback regulation of only one SBEI gene has a minimal effect on the structure of starch (Flipse et al., 1996), although some qualitative changes were identified in another work (Safford et al., 1998). However, in potatoes, inhibition of the combination of SBEII and SBEI genes led to a much larger increase in the relative amylose content than inhibition of SBEII alone (Schwall et al., 2000).

In higher plants, there are two types of branching enzymes, determined by their specificity for the substrate, namely, isoamylase-type branching enzymes and pullulanase-type branching enzymes (Myers et al., 2000). Sugary-1 mutations in corn and rice are associated with a deficiency of both branching enzymes (James et al., 1995, Kubo et al., 1999), but the causative mutation was mapped in the same position as the isoamylase-type branching enzyme gene. Representative starch biosynthesis genes that have been cloned from crops are listed in Table 1.

Table 1
Genes for starch-branching enzymes,
characterized for crops View Isoform SBE Clone Type Reg. No. Literature Corn SBEI cDNA U17897 Fisher et al., 1995 genomic AF072724 Kim et al., 1998a SBEIIb cDNA L08065 Fisher et al., 1993 genomic AF072725 Kim et al., 1998 SBEIIa cDNA U65948 Gao et al., 1997 Wheat SBEII cDNA Y11282 Nair et al., 1997 SBEI cDNA and genomic AJ237897 (SBEI gene)
AF002821 (SBEI pseudogen)
AF076680 (SBEI gene)
AF076679 (SBEI cDNA) Baga et al., 1999
Rahman et al., 1997
Rahman et al., 1999 SBEI cDNA Y12320 Repellin et al., 1997 SBEIIa cDNA and genomic AF338432 (cDNA) Rahman et al., 2001 SBEIIb cDNA and genomic AF338431 (gene) Wo 01/62934 SBEIIb cDNA Wo 00/15810 Rice SBEI cDNA D10752 Nakamura and
Yamanouchi, 1992 SBEI genomic D10838 Kawasaki et al., 1993 RBE3 cDNA D16201 Mizuno et al., 1993 Barley SBEIIa and SBEIIb cDNA and genomic AF064563 (SBEIIb gene)
AF064561 (SBEIIb cDNA)
AF064562 (SBEIIa gene)
AF064560 (SBEIIa cDNA) Sun et al., 1998

Starch is widely used in the food, paper and chemical industries. The physical structure of starch can have a significant effect on the nutritional and technological properties of starch used in the manufacture of food or non-food products or industrial products. Some characteristics can be used as indicators of the structure of starch, including the distribution of amylopectin chain lengths, degree and type of crystallinity, and properties such as gelatinization temperature, viscosity, and swelling volume. Changes in the length of the amylopectin chain can serve as an indicator of changes in crystallinity, gelatinization or degradation of amylopectin.

The composition of starch, and in particular the form called resistant starch, which may be associated with a high amylose content, is important for the normal functioning of the intestine, and in particular the large intestine. Accordingly, high amylose starches have been produced in some cereals, such as corn, for use in foods that can be used as a means of stimulating normal bowel function. The beneficial effects of resistant starch are due to its entry as a nutrient into the large intestine, where the intestinal microflora is provided with an energy source that allows fermentation to form inter alia short-chain fatty acids. These short chain fatty acids provide colonocytes with nutrients, enhance the absorption of certain nutrients by the colon and stimulate the physiological activity of the colon. In general terms, if resistant starches or other dietary fiber does not enter the colon, then it becomes relatively metabolically inactive.

Although chemically or otherwise modified starches can be used to prepare food products that provide functional properties that cannot be provided normally by unmodified sources, such processing tends to either change the valuable qualities of the other components or to impart an undesirable taste due to processes involved in the modification. Therefore, in food products, it is preferable to use sources of components that can be used in unmodified forms.

Every year, all over the world, more wheat is grown than any other grain crop. Known changes in the structure of wheat starch, compared with changes observed in corn or rice, have limitations, partly due to the efficiency of wheat transformation, which occurs with a delay compared to other grains, and also due to the hexaploid nature of bakery wheat. The presence of three genomes in Triticum aestivum has an inhibitory effect due to masking of mutations in individual genomes, in contrast to more easily identifiable mutations in diploid species. Mutations in SBEIIb , corresponding to phenotypes of amylose chain elongation in corn and rice, have not been characterized in wheat. In wheat, the phenotype reported by the SBEIIa and SBEIIb mutations is still unknown. Known mutants are waxy mutants (GBSS, Zhao & Sharp, 1998) and a mutant that completely lacks the SGP-1 protein (Yamamori et al., 2000), produced by crossing lines that lack specific genomic forms A, B and D protein SGP-1 (SSII), as determined by protein electrophoresis. Assessment of seeds not containing SSII showed that this mutation leads to a change in amylopectin structure, to deformation of starch granules and to an increase in the relative amylose content of starch by approximately 30-37%, which is approximately 8% higher than wild-type levels (Yamamori et al ., 2000). Amylose levels are measured by calorimetric analysis, amperometric titration (both of which are based on iodine binding) and the method using concanavalin A. Starch obtained from a mutant that does not contain SSII showed a lower gelatinization temperature compared to starch obtained from an equivalent non-mutant plants. The starch content was reduced by 60% compared to the wild-type plant and lower by less than 50% compared to plants whose grain did not contain SSII.

WO 99/14314 describes the isolation of the SBEIIa gene from Aegilops tauschii , a diploid plant related to wheat, but did not report the production of wheat with modified starch.

WO 00/15810 describes the cloning of wheat SBEIIb gene cDNA. As a result of such cloning, wheat plants with altered amylose levels were not obtained and it was not reported that the obtained wheat had starch containing at least 50% amylose.

In WO 01/62934 also describes a wheat SBEIIb gene and has been proposed to incorporate inhibitors of branching enzyme activity into a wheat plant but has not been reported that wheat having starch comprising at least 50% amylose.

WO 01/32886 describes a cDNA encoding an SBEI form in a wheat endosperm. It has been found that the encoded polypeptide is predominantly associated with A-type starch granules. At the same time, SBEI activity was not suppressed and there was no change in the morphology of starch granules or an increase in the level of amylose in wheat.

It follows that there is currently no data on the production of wheat containing starch with an amylose level of approximately more than 50%. Although high amylose varieties of corn and barley are known, the products obtained from these crops have certain disadvantages compared to high amylose products from wheat, which is the preferred cereal, for example, for baking bread and making pasta (pasta) or noodles.

Although high-amylose starches of these types are valuable products, however, wheat starch with a high amylose content is preferred, especially if it is associated with easier synthesis and other characteristics, such as, for example, less modification after harvest. Such starch products are also relatively degradable and more beneficial to health.

general description

It will be apparent to those skilled in the art that changes and modifications not specifically described herein may be made to the invention described herein. It should be noted that the invention described here includes all of these options and modifications. The present invention also includes all stages, features, compositions and compounds, taken separately or together and related to the present description or specified in the present description, as well as any and all combinations of any of two or more of these stages or features.

Used in the present description, the term “include” and its variants “contains” or “comprising”, unless the context indicates otherwise, means the inclusion of an established whole element or stage, or a group of elements or stages, but does not exclude the presence of any other element or stage, either groups of elements or stages. The scope of the present invention is not limited to the specific options described, which are provided for illustrative purposes only. Obviously, functionally equivalent products, compositions and methods are included within the scope of the present invention described herein.

A detailed bibliography of publications submitted by the applicants in the present description is given at the end of the description. The works cited herein are hereby incorporated by reference in their entireties. Previous work, including any one or more of the preceding documents, should not be construed as confirmation or suggestion, that is, the prior art indicated is only publicly available information known in Australia, or is part of such publicly available information known in Australia.

As used herein, the term “derived from” indicates that a particular element or group of such elements is derived from specific species, however, this does not mean that they must be obtained directly from this particular source.

The designation of the nucleotide residues mentioned here corresponds to the designations recommended by the IUPAC-IUB Biochemical Nomenclature Commission, where A is adenine, C is cytosine, G is guanine, T is thymidine.

Description of the invention

In a first aspect, the present invention relates to grain obtained from a wheat plant, wherein the amylose content of the starch of the grain is at least 50%. This wheat plant may have a reduced level of expression of the SBEIIa gene and activity of the SBEIIa enzyme or both, and preferably a reduced level of expression of both the SBEIIa gene and SBEIIb gene and a reduced level of activity of these enzymes or both. Said grain may include a genetic modification, which is either a mutation of the SBEIIa gene, which inhibits the expression of the SBEIIa gene, enzyme activity, or both, or an integrated nucleic acid that inhibits the expression of the SBEIIa gene, enzyme activity, or both. In addition, the indicated grain may contain a similar genetic modification in SBEI. Such a grain may also have an altered level of protein and / or activity of an enzyme selected from the group consisting of ADP-glucose pyrophosphorylase, GBSS, SSI, SSII, SSIII, a branching enzyme such as isoamylase, and a branching enzyme such as pullulanase. Such a grain may contain a transgene, and this transgene may encode an antisense, co-suppressor, ribozyme or duplex RNA molecule. The presence of such a transgene preferably leads to a decrease in the expression level of mRNA encoding SBEIIa. The specified grain may contain a mutation in the SBEIIa gene, and in one embodiment, it may contain a silent mutation (null mutation) of the SBEIIa gene in at least one genome, and may contain a silent mutation of the gene in two or three genomes. The amylose content in the starch of the indicated grain can be at least 40%, 50%, 55%, 60%, 70% or 80%. In another embodiment, at least 50% of the starch granules in the grain are non-birefringent when observed in polarized light. Said grain may be non-wrinkled, and its average weight may be at least 36 mg or 40 mg. Alternatively, the pure starch content is at least 25% (w / w) or at least 35% (w / w), while the starch content in wild-type grain may be at least 90%. This grain may be whole, husked, ground, crushed, crushed, crushed, crushed, crushed or scorched.

In another embodiment of its first aspect, the present invention relates to a grain obtained from wheat, where said grain contains starch and its genetic modification, which leads to a decrease in the expression level of the SBEIIa gene, the activity of the enzyme SBEIIa in the endosperm, or both, compared to the grain wild type, and which contains a mutation of the SBEIIa gene or an integrated nucleic acid encoding an SBEIIa gene expression inhibitor, wherein the amylose content of the starch of said grain is at least 30%.

In a second aspect, the present invention relates to a grinding product obtained from a grain of the first aspect of the invention, including, but not limited to, flour, whole grain flour, cereal or starch, obtained from grain of the present invention, or food products comprising said flour , whole grain flour, cereals or starch, or rolled out, rolled or pressed cereal products. Such a product may be flour, whole grain flour, cereal or starch, obtained from grain of the first aspect of the present invention, mixed with flour, whole grain flour, cereal or starch obtained from another source.

In a third aspect, the present invention relates to starch granules or to starch obtained from wheat grains of the first aspect of the invention. In a specific embodiment of the third aspect of the invention, the wheat plants further have a reduced level of activity of the enzyme SBEIIa in the endosperm.

In its fourth aspect, the present invention relates to a composition comprising starch of the third aspect of the present invention and food ingredients or water. This aspect includes food products and non-food compositions and a mixture of starch with other types of starch or starch-containing products.

In its fifth aspect, the present invention relates to a composition comprising starch granules of the fourth aspect of the invention described above, and another food ingredient or water.

In its sixth aspect, the present invention relates to a wheat plant, which can be used to produce grain or starch granules or starch in accordance with the previous aspects. Such a wheat plant may be transgenic or non-transgenic, or it may produce transgenic or non-transgenic grain.

In its seventh aspect, the present invention relates to a method for growing a wheat-producing wheat plant, wherein said method comprises the steps of: i) introducing a genetic modification into the parent wheat plant or its grain, and ii) identifying the progeny of the plant or seeds of the parent wheat plant or its seeds that have a reduced level of expression of the SBEIIa gene, the activity of the SBEIIa enzyme in the endosperm, or both compared to plants or seeds of the wild type, where the specified grain contains starch, and where obsession amylose in said starch of the grain is at least 50%.

In a second embodiment of its seventh aspect, the present invention relates to a method for growing a grain producing wheat plant, wherein said method comprises the steps of: i) introducing a genetic modification into a parent wheat plant or into its grain, wherein said genetic modification comprises a SBEIIa gene mutation or an integrated nucleic an acid encoding an SBEIIa gene expression inhibitor, and ii) identifying a progeny of a plant or seeds of a parent wheat plant or seeds thereof that have a reduced expression level this gene SBEIIa, the activity of the enzyme SBEIIa in the endosperm or both compared with plants or seeds of the wild type, where the specified grain contains starch, the amylose content of which is at least 30%. Said step of introducing a genetic modification may include administering an exogenous nucleic acid expressing an SBEIIa gene expression inhibitor, or mutagenesis of a parent wheat plant.

In a third embodiment of its seventh aspect, the present invention relates to a method for growing a wheat-producing wheat plant, wherein said method comprises the steps of: i) introducing a genetic variant into a parent wheat plant or into its grain, where said genetic modification contains a mutation of the SBEIIa gene, and ii ) identification of the progeny of the plant or seeds of the parent wheat plant or seeds that have a reduced level of expression of the SBEIIa gene, activity of the SBEIIa enzyme in the endosperm, or both compared to wild type, iii) introducing the genetic modification into the parent wheat or grain plant, where the genetic modification contains a mutation of the SBEIIb gene, and iv) identifying the progeny of the plant or grain of the parent plant or wheat grain having a reduced expression level of the SBEIIb gene, SBEIIa enzyme activity endosperm, or both compared to wild-type grain; v) crossing a plant having a reduced level of expression of the SBEIIa gene, activity of the SBEIIa enzyme in the endosperm or both with a plant having a reduced level of expression of the SBEIIb gene, activity of the SBEIIb enzyme in the endosperm, or both; and identifying a wheat plant having a reduced level of gene expression, activity of the SBEIIa and SBEIIb enzymes, as well as both.

In a fourth embodiment of its seventh aspect, the present invention relates to a method for cultivating a wheat plant having a relative amylose content in grain starch of at least 50%, and preferably having a reduced activity of the SBEIIa enzyme in the endosperm, wherein said method comprises: a) identification wheat plants or grains having a reduced activity of the SBEIIa enzyme expressed from the wheat genome A, B or D; and b) crossing said wheat plant or wheat plant grown from grain of step (a) with a second wheat plant having a reduced activity of the SBEIIa enzyme; or c) crossbreeding a plant having a reduced activity of the SBEIIa enzyme with a wheat plant having a reduced activity of the SBEIIb enzyme; and identifying a wheat plant having reduced activity of SBEIIa and SBEIIb enzymes. A preferred plant of the seventh aspect is Triticum aestivum ssp. aestivum .

In its eighth aspect, the present invention relates to a method for producing modified starch, comprising modifying a plant by the method defined above and extracting starch having altered properties.

In its ninth aspect, the present invention relates to a method for identifying a wheat plant or its grain by mutation in the SBEIIa gene or SBEIIb gene, where the method includes the steps of screening a population of wheat plants or its grain using a molecular marker attached to the SBEIIb gene, or wheat SBEIIa gene, respectively, and to a method for identifying a plant or grain for the presence or absence of said attached molecular marker.

In a second embodiment of its ninth aspect, the present invention relates to a method for identifying a wheat plant or its grain for a mutation in the SBEIIa gene or SBEIIb gene, where the method includes the steps of screening a wheat plant population or its grain using an antibody that is specific for SBEIIb protein or wheat SBEIIa protein, respectively, and a method for identifying a plant or grain for the presence or absence of binding to an antibody.

In its tenth aspect, the present invention relates to a grain obtained from a wheat plant and containing a mutation, where the SBEIIa gene is absent in the long arm of chromosome 2A, or where the SBEIIa gene present on the long arm of chromosome 2A contains a mutation that leads to a decrease in the level of SBEIIa protein , activity of the SBEIIa enzyme, or both, in the endosperm of said grain compared to wild-type grain. Such a mutation may be a null mutation of the SBEIIa gene, or it may be a deletion of at least part of the SBEIIa gene. The indicated grain may also contain a mutation where the SBEIIb gene is absent in the long arm of chromosome 2A, or where the SBEIIb gene present on the long arm of chromosome 2A contains a mutation that leads to a decrease in the level of SBEIIb protein, SBEIIb enzyme activity, or both the endosperm of the indicated grain compared to wild-type grain. Deletion can stop the expression of the SBEIIa and SBEIIb genes on the long arm of chromosome 2A.

The indicated plant may be a durum durum wheat plant, which may additionally contain a genetic modification, leading to a decrease in the activity of the starch-branching enzyme encoded by the SBEIIa gene on the long arm of chromosome 2B compared to wild-type grain. Another genetic modification may be a deletion of the SBEIIa gene on the long arm of chromosome 2B or a mutation of the SBEIIa gene on the long arm of chromosome 2B, resulting in a decrease in the activity of the SBEIIa enzyme in the endosperm of the indicated grain compared to wild-type grain.

The plant may be Triticum aestivum ssp. aestivum ,, which probably additionally contains a genetic modification, leading to a decrease in the activity of the starch-branching enzyme encoded by the SBEIIa gene on the long arm (s) of chromosome 2B, chromosome 2D or both chromosomes, compared with wild-type grain. Another genetic modification may be a deletion of the SBEIIa gene in at least one of these chromosomes, or a mutation of the SBEIIa gene in at least one of these chromosomes, which lead to a decrease in the activity of the SBEIIa enzyme in the endosperm of this grain compared to wild type.

This plant may have a built-in nucleic acid encoding an inhibitor of SBEIIa gene expression, its activity, or both. The level of activity of the SBEIIa enzyme can be reduced by at least 40% compared with the level of enzymes in wild-type grain. The amylose content in the starch of such a grain may be at least 30%, or at least 50%. Said grain may be non-wrinkled and its average weight may be at least about 36 mg. At least 50% of the starch granules in said grain can be non-birefringent when observed in polarized light. In one embodiment of the invention, the starch content of the refined grains is at least 25% (w / w) or at least 90% of the starch content of the wild-type grain.

Amylopectin present in the grain according to any embodiment of the present invention may contain a reduced level of dp (D-pyranose) fraction with a long chain of 4-12 units, compared to wild-type amylopectin, as measured after amylopectin isoamylase was decomposed.

In addition, said grain may contain reduced levels of SBEI protein, SBEI enzyme activity, or both, and may also contain altered levels of the enzyme compared to wild-type grain, where said enzyme is selected from the group consisting of ADP-glucose pyrophosphorylase, GBSS, SSI , SSII, SSIII, a branching enzyme of the isoamylase type and a branching enzyme of the pullulanase type, and any combination thereof.

Embodiments of the tenth aspect of the present invention encompass grain; starch granules extracted from grain; as well as products produced from this grain or its starch, such as, for example, flour, whole grain flour or cereals.

A brief description of the graphic material

Figure 1 . The sequence of the starch-branching enzyme IIa gene ( wSBE II-D1) [SEQ ID NO: 1] from A. tauschii , corresponding to the SBEIIa gene of the hexaploid wheat genome D ( T.aestivum) .

Figure 2 . Incomplete SBEIIb gene sequence ( wbe2b genomic sequence ) [SEQ ID NO: 2] from T.aestivum .

Figure 3 . Schematic design of duplex RNA. A. Gene elements are arranged in the following order: promoter, SBEIIa or SBEIIb gene sequence (exons 1, 2, and 3) in sense orientation, intron (intron 3), SBEIIa or SBEIIb gene sequence (exons 1, 2, 3, and 4) antisense orientation, and transcription / polyadenylation terminator sequence. B. The transcript of the ds-SBEIIa and ds-SBEIIb genes forms a “hairpin” RNA structure with a double-stranded region formed by hybridization between sense and antisense sequences. The intron sequence limited to the nucleotides GT and AG is excised.

Figure 4 . Starch granules observed under an optical microscope and derived from (A) wheat seeds with wild-type starch granules derived from ds-SBEIIa transgenic line 83.1b, (B) wheat seeds with deformed wild-type starch granules derived from ds-SBEIIa- transgenic line 50.1b.

Figure 5 . Birefringence of starch granules derived from wheat seeds shown in FIG. 4 and visualized in polarized light.

6. Comparison of incomplete wheat SBEIIa cDNA sequences. sbe 9 corresponds to part AF338432.1. The following partial sequences are shown: Y1182 [SEQ ID NO: 3], sr997 [SEQ ID NO: 4], sr995 [SEQ ID NO: 5], sbe9 [SEQ ID NO: 6].

7 . Comparison of incomplete wheat SBEIIa sequences for the first 63 amino acids using the PILEUP program. The alleged localization of the genome in the genes corresponding to clones is indicated.

Fig . 8 . Comparison of the deduced amino acid sequences of the D-genome polypeptide (sr854) [SEQ ID NO: 7] with genome A or B products (y11282) [SEQ ID NO: 8]. The transferred sequence (positions 1-54) is shown in italics.

Fig.9 . PCR amplification of the intron 3 region of the SBEIIb gene for various wheat lines (lanes 1-11) using ARA19F and ARA23R primers after hydrolysis with Rsa 1. The bands corresponding to genomes A, B and D are shown by arrows. Lane 3 (Aus17340) and lane 5 (Aus10103) lack a specific marker of genome D, and lane 8 (Aus12509) and lane 9 (Aus12565) lack a marker of genome B.

Figure 10. Southern hybridization of Hind III-hydrolyzed DNA of wheat lines using a probe from the intron 3 region of the SBEIIb gene. Tracks correspond to: 1) Aus12565, 2) Aus12509, 3) Aus10103, 4) CSDT2DL-4, 5) Aus12530 (durum wheat), 6) CSDT2BL-9, 7) Aus6323, 8) CSDT2DS, 9) Aus17340, 10) Aus12745 11) CSDT2DL-4; 12) Aegilops tauschii .

11 . Screening of the F2 cross population of Aus17340a X Aus12509 by PCR amplification of the intron 3 region of the SBEIIb gene using AR2b19cF AR2b23cR primers followed by Rsa I cleavage. Track 8 lacks genomic markers B and D, and therefore the BD54 line is a BD line that lacks two genome B and D.

Fig . 12 . Southern hybridization of Hind III- (lanes 1-4) and EcoR1- (lanes 5-8) -hydrolyzed BAC clones using a probe from the intron 3 region of the SBEIIb gene. The tracks correspond to: 1) BAC4, 2) BAC5, 3) BAC9, 4) BAC12, 5) BAC4, 6) BAC5, 7) BAC9, 8) BAC12.

Fig.13 . A) FISH using a wSBEII-DA1 probe and a probe with a repeating DNA sequence (pSc 119.2) for A. tauschii chromosomes (main photograph and lower insert) and for wheat chromosomes (upper insert). C) FISH probe SBEIIb for wheat chromosomes.

Fig.14 . SDS-PAGE analysis of grain-bound proteins in wild-type Chinese spring wheat (Chinese Spring) (СS) and in wheat lines not containing SGP-1, carried out at several indicated stages of seed development (10, 15, 25 days after flowering , M = mature). The intensity of a strip of protein visualized in a silver stained gel was measured. The intensity of the HBSS band in mature CS seeds was normalized by dividing by 100, and the number of other enzymes produced in this developmental stage was expressed as the percentage of HBSS in mature wheat CS. a) GВSS, b) SSI, c) SBEII. Black bars indicate the absence of SGP-1. An example of a gel electrophoretogram for grain bound CS proteins and a line containing no SGP-1 is presented.

Fig.15 . Relative amounts of SBEIIa and SBEIIb in the soluble fraction. SDS-PAGE immunoblots were scanned and protein band intensity was measured by visualization. Protein amounts were estimated based on SBEIIa- and SBEIIb-fusion proteins used on gels as a standard.

Fig.16 . A. Anion-exchange chromatography of the activity of a branching enzyme in wheat endosperm (Rosella cultivar). Soluble proteins in the endosperm were fractionated using ammonium sulfate and chromatographed on a Sephacryl S-200 column, and then loaded onto a Resource Q anion exchange column. B) The analysis was performed by immunodetection of the SBEI protein in wheat endosperm, separated by electrophoresis in non-denaturing PAAG, using anti-WBE1 antibodies. The SBEI protein bands labeled A and B are products originating from genomes A and B, respectively, and Di and Dii are products originating from genome D. Lanes correspond to extracts from: 1, CS; lane 2, N7BT7A; lane 3, N7AT7B; lane 4, N7DT7A. C) Analysis of the purified fractions representing the active peaks in the anion exchange chromatogram was carried out by immunodetection using an anti-WBE1 antibody. Lane 1, crude soluble endosperm extract; lane 2, fractions representing peak 1; lane 3, fraction representing peak 2.

Fig.17 . Screening of double haploid progeny from VC3.1.11 x CS7AL-15 crosses for segregation of SBEI isoforms was performed by immunodetection using an anti-WBEI antibody. Lanes 1-14 correspond to lines of double haploid offspring. Lane 6 shows the SBEI triple null mutant line, designated A113, and lane 7 shows the line normal for SBEI isoforms and designated D28.

18. PCR amplification of DNA from gamma-induced mutant seeds (lanes 1-6) of the Veery 3 x Gabo 1BL.1RS crosses using AR2b19cF / AR2b23cR primers. Lane 2 shows the mutant seeds of MLT2B8, and Lane 7 shows the Chinese Spring variety.

Fig.19 . PCR amplification of DNA from wheat lines using genome A specific primers for the SBEIIa wheat gene, ARIIaAF / ARIIaAR. Lanes 1-5 correspond, in order: CS, MLT2B8, MLT2D1, Dt2AS and BD219 (a plant that is a null mutant for both SBEIIa and SBEIIb genes in both B and D genomes).

Fig.20 . Chromatogram on a 2B gel with Sepharose CL for starch of wheat lines a) Ass144008 and b) Ass 144087 analyzed using a starch analysis kit (Sigma).

Fig.21 . Comparison of the chain length profile of starches from transgenic wheat lines with a non-transformed control, HB1 (wheat). The percentage of the total mass of individual starch oligosaccharides from the non-transformed control was subtracted from the corresponding values for starch from transgenic lines. Samples: 085 (♦), 025 (▲), 008 (O).

DETAILED DESCRIPTION OF THE INVENTION

Modification of SBEIIa in Wheat

The present invention is based on the discovery that SBEIIa activity in wheat endosperm leads to the production of modified starch, and in particular, relatively high levels of amylose in wheat grain. This unexpected result contrasts with the results obtained for corn and rice, where the mutation in SBEIIa did not change the profile of amylopectin / amylose (Blauth et al., 2001, Nakamura, 2002). In another embodiment, the present invention includes a modification of one or more additional activities of an enzyme involved in starch biosynthesis, such as a decrease in SBEIIb and SBEIIa activity. A mutation of the genes encoding these two activities was created due to the unexpected discovery of the fact that in wheat, unlike corn and rice, SBEIIa and SBEIIb are directly linked to each other. The authors also unexpectedly found that the grain of a wheat plant, which has reduced levels of activity of SBEIIa and SBEIIb, is not wrinkled.

A method of obtaining a wheat plant

In one of its aspects, the present invention relates to a method for producing a wheat plant containing modified starch in its grain, and in particular, an increased level of amylose in starch by at least 30%. Typically, in hexaploid wheat and durum wheat, the amylose content in starch is from about 18 to about 30%, and in some mutants (deficient in SGP-1) to about 35%. In one embodiment of the invention, the method of the present invention includes the step of introducing a genetic modification into a parent wheat plant or its seeds in order to produce wheat plants in the grain of which starch will contain at least 30% amylose. In the present invention, the amylose content of starch is determined by weight (w / w), that is, the mass of amylose is expressed as a percentage of the mass of starch by weight of grain. In other embodiments of the invention, the amylose content of starch is at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% (each - mass / mass). In other embodiments of the invention, the method allows to achieve an amylose content of at least 80% or at least 90% (w / w).

In another embodiment of the invention, said method comprises altering, preferably reducing the level of protein, namely, starch-branching enzyme IIa (SBEIIa) or enzymatic activity, or both, in the wheat endosperm. That is, the genetic modification that is introduced into the wheat plant leads, directly or indirectly, to a change in the SBEIIa level, and hence to the starch modifications described in this application. In another embodiment of the invention, which does not exclude the previous option, the method involves changing, and preferably, reducing the expression level of the SBEIIa gene in wheat endosperm, or it involves introducing a mutation of the SBEIIa gene in wheat, which leads to a decrease in SBEIIa activity in the endosperm. Decreased expression of the SBEIIa gene or other genes can be achieved by embedding a nucleic acid, for example a transgene that encodes an inhibitory molecule. Examples of inhibitory molecules are antisense, co-suppressor, ribozymes, or duplex RNA molecules.

As used herein, the terms “change”, “increase”, “increased”, “decrease”, “reduced”, “inhibited”, or the like are considered as semantically related concepts compared to the concepts of “wild type” or “unmodified”. The term “protein level” means the amount of a particular protein, for example SBEIIa, which can be measured by any method known to those skilled in the art, such as, for example, Western blot analysis or other immunological methods. The term “level of enzymatic activity” means the amount of a particular enzyme measured in an enzymatic assay. It should be noted that the level of enzyme activity in the mutant can be changed, but not the level of expression (quantity) in the protein itself. Conversely, the amount of protein can be changed, and its activity can remain at the same level if more or less active protein is produced. The amount and activity may also decrease if, for example, the gene encoding the enzyme is inactivated. In some embodiments of the invention, the reduction in protein or activity is at least 40%, or at least 60%, or at least 75%, at least 90%, or at least 95% by compared with the level of protein or activity in the endosperm of unmodified wheat. A decrease in protein level, enzymatic activity, or gene expression can occur at any stage of grain development, and in particular, at the stage of grain loading, when starch is synthesized in the developing endosperm, or at all stages of grain development, up to its ripeness.

As used herein, the term “starch” is defined as a polysaccharide consisting mainly of α-glucopyranose units. Starch is the main storage carbohydrate of wheat, is synthesized in amyloplasts and is formed and accumulates in starch granules. It includes amylose, mainly a linear α-1,4-D-glucopyranose polymer and amylopectin, which has short chains of α-D-glucopyranose units, linked mainly by α-1,4-D-bonds with α- 1,6-affiliated branches. Wild-type wheat starch contains about 20-30% amylose and about 70-80% amylopectin. Another significant difference between the two molecules is molecular weight. Amylose has a helical conformation with a molecular weight of 10 ⁴ -10 ⁶ , and amylopectin has a molecular weight of 10 ⁷ -10 ⁸ Daltons. Recent studies have shown that approximately 0.1% of the α-1,6-glycosidic branch points can be present in amylose, and therefore it is defined as being basically a “linear molecule”. The term “amylose” is defined herein as a substantially linear molecule consisting of α-1,4-linked glucoside (glucopyranose) units and amylose-like long chain amylopectin (sometimes called “intermediate material” or “amylose-like amylopectin”, Takeda et al., 1993b; Fergason, 1994). Amylose content can be determined by any methods known to those skilled in the art, including size exclusion HPLC performed, for example, by methods using 90% (w / v) DMSO and concanavalin A (Megazyme Int., Ireland), or, preferably, by iodometric methods, for example, as described in Example 1. The HPLC method may include (Batey & Curtin, 1996), or may not include, starch branching. Based on the grain mass and amylose content, it is possible to calculate and compare the amounts of amylose accumulated in the grain of transgenic and control lines.

In another embodiment of the invention, said method comprises the step of determining the amount of SBEIIa activity in the wheat endosperm by any known method. In some embodiments of the invention, the protein level is measured, for example, by immunodetection methods such as Western blot analysis or ELISA analysis, or its mRNA level is measured in accordance with well known methods such as Northern blot hybridization analysis or polymerase chain reaction involving reverse transcriptase (RT-PCR). In another embodiment of the invention, said method comprises the step of selecting or screening a wheat plant or grain thereof having an altered level of SBEIIa protein or enzymatic activity in the endosperm. The selection step can be carried out on the basis of a reduced level of SBEIIa activity or protein level, or it can be carried out based on the phenotype of a wheat plant grain, such as an increased content of amylose or a reduced content of amylopectin, or based on a visual assessment of the phenotype, for example, wrinkled grain or on the modified properties of starch granules.

It should be noted that the present invention includes a method for identifying a wheat plant with altered starch properties in grain by any of the methods described herein, or directly or indirectly determining properties, for example, by detecting the presence of a genetic modification in a plant or in its grain. Such a plant may be a plant belonging to a population of wheat plants, for example, selection wheat.

SBE activity can be measured directly by enzymatic analysis, for example, by stimulation analysis (Boyer & Preiss, 1978). This assay measures SBE stimulation of the incorporation of methanol insoluble glucose-1-phosphate into the polymer (α-D-glucan) by phosphorylase a . SBE activity can be determined using an iodine staining assay, which measures the decrease in optical density of the glucan-polyiodine complex due to branching of glucan polymers. SBE activity can also be assessed using branch-link analysis, which assesses the generation of reducing ends from reduced amylose as a substrate after isoamylase cleavage (Takeda et al., 1993a). Preferably, said activity is measured in the absence of SBEI or SBEIIb activity. The SBE isoforms exhibit different substrate specificities, for example, SBEI has a higher amylose branching activity, while SBEIIa and SBEIIb contribute to the acceleration of branching when reacted with an amylopectin substrate. These isoforms can also be identified based on the length of the transferred glucan chain. The SBE protein can also be evaluated using specific antibodies, such as the antibodies described herein. SBEII activity can also be measured during grain development in the developing endosperm, or alternatively, in mature grain, where the protein is still present in equivalent but unmodified grain and can be analyzed by immunological methods.

In another aspect, the present invention relates to a method for altering, and preferably reducing, the activity of a plurality of starch biosynthesis enzymes in wheat endosperm, where one of these enzymes is SBEIIa, so that the amylose content of the grain starch is at least 50% . In some embodiments of the invention, the levels of SBEIIa and SBEIIb proteins or enzymatic activities are reduced, or the levels of all three enzymes SBEIIa, SBEIIb and SBEI are reduced. Other starch biosynthesis enzymes that can be modified in combination with SBEIIa are SSI, SSII, SSIII. Starch-branching enzymes can also be modified, for example, with respect to the activity of isoamylase or pullulanase. The present invention also relates to any combination of the above enzymes, provided that SBEIIa is modified. In another embodiment of the invention, the activity of one or more starch biosynthesis enzymes in plant tissues other than endosperm is altered; for example, in leaves, SBEI or SBEII activity can be increased to compensate for some loss of activity caused by the influence of a transgene encoding an SBEIIa inhibitory molecule, primarily involved in expression in the endosperm. Such a change may be an increase or decrease in the amount or a change, for example, in expression time. Alternatively, starch synthesis can also be enhanced by overexpression of one or more starch biosynthesis enzymes in combination with a decrease in SBEIIa. Genes encoding such enzymes can come from any type of source, for example, from bacterial or other non-wheat sources, and they can be modified to change the catalytic properties, for example, to change the temperature dependence of the action of enzymes (e.g. see WO 94/09144).

A high amylose phenotype can be achieved by partially or completely inhibiting the expression of the SBEIIa gene or SBEIIa and SBEIIb genes . The degree of inhibition of such a gene or genes will to some extent determine the properties of starch formed in wheat grain. The nature and degree of modification of SBEIIa- and / or SBEIIb activity can be detected by any gel electrophoresis method used to analyze proteins extracted from modified wheat endosperm. Such a modification may be a decrease in SBEIIa- and / or SBEIIb activity, complete elimination of enzymatic activity, or a change in the distribution of SBEIIb or other enzymes in the endosperm. To conduct these analyzes, starch can be extracted from the endosperm of wheat, and the proteins present in it can be analyzed, for example, as described by Rahman et al., 1995. Well-known methods such as SDS-PAGE and immunoblotting are performed on soluble and granular starch fractions, and the results are used to identify plants or grains that carry a modification of the SBEIIa and / or SBEIIb enzymes.

Wheat plants

In another aspect, the present invention relates to a wheat plant capable of producing grain in which the amylose content of starch is at least 30%. In other embodiments of the invention, the amylose content of starch is at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 80%. In another embodiment of the invention, the wheat plant, the grain of which contains any levels of amylose in starch, has a genetic modification that leads to a decrease in the expression level of the SBEIIa gene, the activity of the SBEIIa enzyme in the endosperm, or both compared to wild-type grain. In a preferred embodiment of the invention, the genetic modification comprises a mutation of the SBEIIa gene or an introduced nucleic acid encoding an SBEIIa gene expression inhibitor. Such an inhibitor may contain an antisense, co-suppressor, ribozyme or duplex RNA molecule or a similar molecule that inhibits the expression and / or activity of SBEIIa .

The wheat plant used here is any plant of the genus Triticum , which are commercially valuable varieties, including, for example, Triticum aestivum L.ssp . aestivum (wheat or bakery wheat), other subspecies of Triticum aestivum, Triticum turgidum L. ssp . durum ( durum wheat, also known as amber durum or durum wheat), Triticum monococcum L.ssp . monococcum (cultivated odnozerenka or spelled), Triticum timopheevi ssp . timopheevi, Triticum turgigum L. ssp. dicoccon (spelled-emmer) and other subspecies of Triticum turgidum (Feldman). Such wheat may be hexaploid wheat having the AABBDD type genome, or tetraploid wheat having the AABB type genome. Since the genetic modification in wheat of the present invention can be transmitted to some related species, including rye and barley by hybridization, the present invention also encompasses hybrid species so derived, including the triticale variety, which is a hybrid of wheat and rye. In a particular embodiment, such a wheat plant is a view of Triticum aestivum, and preferably subspecies aestivum. Alternatively, since mutations or transgenes can be easily transferred from Triticum aestivum to durum wheat, Triticum turgidum L. ssp. Is the preferred wheat. durum .

The present invention also relates to wheat plants with a reduced level of SBEIIa protein, enzymatic activity in the endosperm, or both, wherein said wheat plant is capable of producing grain whose starch has an increased amylose content compared to starch extracted from a wild-type plant. A decrease in the level of SBEIIa can occur, at least during part of the process of grain development or during the whole process of plant development until it reaches maturity. In another embodiment of the invention, the level of SBEIIa in the endosperm is reduced by at least 50%, at least 75%, at least 90%, or at least 95% compared with the level in the wild plant type. The term “wild type” is used in its usual meaning known to geneticists and includes varieties or genotypes of wheat that are not modified as described in this application.

The present invention also relates to progeny of plants and to grain, which retains the desired genotypic and / or phenotypic properties of parent wheat plants. The present invention also relates to any material for growing wheat plants that can be used to cultivate plants with desired properties, such as cultured tissues or cells.

The present invention also relates to wheat plants which, in addition to reduced SBEIIa activity, contain altered, and preferably reduced, levels of SBEIIb enzymes or other starch biosynthesis enzymes. Plants with reduced SBEIIa and SBEIIb activity can be produced by crossing a plant having a reduced level of SBEIIa with a plant having a reduced level of SBEIIb, or by introducing a transgene encoding a molecule that inhibits the expression of both SBEIIa and SBEIIb genes . Due to the close linkage of the SBEIIa and SBEIIb genes in wheat, as was revealed in the present invention, plants with reduced activity of both enzymes can also be produced by identifying varieties that do not contain isoforms of SBEIIa and SBEIIb encoded by one of the wheat genomes and by crossing such varieties for producing a plant containing a reduced level of isoforms encoded by at least two genomes.

The present invention also relates to genetic (s) change (s) or mutations in other genetic phenotypes or in other species that can be hybridized with the wheat plant described above. Altered (mutant) plants can be crossed with plants that have a more desirable genetic background. After the initial crossing, a suitable number of backcrosses can be obtained to remove a less desirable background. The desired genetic background may include a suitable combination of genes providing yield in industrial production and other characteristics such as agronomic efficacy or resistance to abiotic stress. The genetic background may also include other changes in starch biosynthesis or modification of genes, for example, genes from other wheat lines having a wrinkled endosperm whose causal gene is unknown.

Such plants may be transgenic or non-transgenic.

The present invention also relates to wheat plants containing a mutation where the SBEIIa gene is absent in the long arm of chromosome 2A (2AL), or where the SBEIIa gene present on the long arm of chromosome 2A carries a mutation leading to a decrease in the level of activity of the SBEIIa enzyme in the endosperm of the indicated grain compared to wild-type grain. Despite the wide screening of a library of 2400 wheat varieties, the authors did not find such plants in nature, which suggests that nature may select for the conservation of the functional SBEIIa gene on chromosome 2AL. However, these plants can be produced and identified after mutagenesis. Such plants are non-transgenic plants, which in some cases are preferred for sale. These plants may be bakery wheat, durum wheat or other wheat varieties. In a preferred embodiment of the invention, the wheat plant contains on the chromosome 2AL a deletion of at least a portion of the SBEIIa gene, which can extend to at least a portion of the SBEIIb gene. As is apparent to every person skilled in the art, hexaploid wheat, such as bakery wheat, has three genomes, which are usually denoted by genomes A, B and D, and tetraploid wheat, such as durum wheat, contains two genomes, usually denoted by genomes A and B. Each genome contains 7 pairs of chromosomes that can be observed by cytological methods during meiosis. Chromosomes are usually designated according to their size, from the largest to the smallest, and therefore chromosome 2 is the second largest, after the largest, chromosome in each genome. Each chromosome has a centromere, which is located asymmetrically on chromosome 2, and therefore the two arms of chromosome 2 are called “short” and “long”. As used herein, the term “long arm of chromosome 2A” has its usual meaning, that is, is defined as the region of the chromosome located between the centromere and the end of the long arm. The terms “long arm of chromosome 2B” and “long arm of chromosome 2D” have the same meaning, except that they refer to chromosome 2 of the wheat genomes B or D, respectively.

The authors found that the SBEIIa and SBEIIb genes are closely linked on chromosome 2 of wheat. In a specific embodiment of the invention, the wheat plant contains many (> 50%) 2AL, where the arm of this chromosome contains a mutation of at least the SBEIIa gene. That is, basically, there is a 2AL chromosome containing a mutation of at least the SBEIIa gene in genome A. The presence of 2AL can be determined by cytological methods, such as, for example, in situ hybridization methods (see example 9), or by methods with using 2AL-specific molecular markers. In a preferred embodiment of the invention, the wheat plant is homozygous for said mutation. The indicated mutation may be a silent mutation (null mutation). Such a mutation may be a deletion.

In a specific embodiment, the deletion allele is derived from plants of MLT2B8 or MLT2D1. Since in these plants the mutant SBEIIa alleles are located on chromosome 2AL, these alleles can be introduced into plants of varieties of bakery wheat or durum wheat by crossing, and therefore such plants, as well as grain and starch products obtained from it, are also included in the scope of the present invention. These alleles can be combined with other genes or alleles responsible for starch biosynthesis, or with other alleles possessing valuable genetic traits.

Obviously, the present invention also relates to methods for producing or identifying such plants of wheat or grain produced by such plants.

Corn

The present invention also relates to wheat grain containing modified starch compared to starch extracted from wild-type wheat grain. As used herein, the term “grain” means mainly ripe grain. This term includes grain harvested from industrial cultivation. In one embodiment of the invention, the modified starch is produced, at least in part, as a result of a decrease in SBEIIa activity in the development of endosperm of wheat grain. In another embodiment of the invention, which does not exclude the previous embodiment, said grain has a high amylose content (percentage of total starch). This amount can be defined as a reduced amylopectin content in starch compared to its content in wild-type plant grain. Wild-type wheat starch contains approximately 20-30% amylose and 70-80% amylopectin. The grain of the present invention contains starch, preferably having at least 50% (w / w) amylose. In another embodiment of the invention, SBEIIa- and SBEIIb activities also decrease during endosperm development. In another embodiment, SBEI activity is also reduced. In other embodiments of the invention, the amylose content of grain starch, measured by methods well known in the art, is at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, or at least 90% (each value is given by weight). An increase in amylose levels can be observed with an abnormal morphology of starch granules or in the absence of birefringence of these granules, observed under an optical microscope or other known methods. In a particular embodiment of the invention, the amylose content is measured by the iodometric method, which may be, for example, a spectrophotometric method, such as, for example, the method described by Morrison & Laignelet (1983), or high performance liquid chromatography (HPLC, e.g., Batey & Curtin, 1996) .

In other embodiments of the invention, the wheat grain contains starch, which has altered physical properties, such as, for example, increased or decreased gelatinization temperature, altered swelling properties during or after gelatinization, altered viscosity, altered distribution of chain lengths in amylopectin, or any combinations. An increased or decreased gelation temperature may be characteristic of the first gelation peak, the second gelation peak, or both peaks. One or more starch properties, such as, for example, gelatinization enthalpy, may remain unchanged. The temperature of the first peak (maximum) gelatinization, measured using differential scanning calorimetry, can be increased by at least 3 ° C or 5 ° C, and preferably at least 7 ° C or 8 ° C. and more preferably, at least 10 ° C over the temperature of the first peak for the corresponding starch of wild-type grain. In a specific embodiment of the invention, the temperature rises from 3 to 12 ° C.

The grain may be wrinkled or wrinkled, and preferably, it is wrinkled. As used herein, the term “non-wrinkled” refers to a large amount of grain, preferably at least 90% of the individual kernels exhibiting a “completed” or fully poured phenotype. This phenotype is usually associated with a normal level or close to a normal level of starch accumulation. In contrast, the term “wrinkled” phenotype, as used herein, means a grain phenotype, where most grains, preferably at least 90% of the grains, have a phenotype with reduced starch accumulation. A slightly wrinkled grain means a decrease in starch content by an average of at least 30%; moderately wrinkled grain means a reduction in starch content by an average of at least 50%; and a highly wrinkled grain means a decrease in starch content by an average of at least 70% compared to wild-type grain. Wrinkling can also be determined by the relative starch content as a percentage by weight of mature grain. The starch content of unmodified wheat grain grown in the field is approximately 65%, and the starch content of wrinkled grain is reduced to less than 50%.

In other embodiments of the invention, the grain has an average weight of at least 36 or 40 mg. The average grain mass is determined by measuring the mass of a known number of wheat grains constituting a representative sample of the grain batch, and dividing the total mass by the number of grains. It should be noted that the characteristics of this grain, such as starch content, average weight and non-wrinkled phenotype, which are similar to the characteristics of wild-type grain, are desirable for industrial grain production.

The present invention also relates to flour, flour products, dough and other products made from or using said grain. This grain may be unprocessed or processed, for example, by fractionation or bleaching. The present invention also relates to wheat grains used for the manufacture of food products derived from the wheat plants of the present invention. In addition, the present invention includes grain processed by other methods, as a result of which this grain can be ground, crushed, rolled, crushed, crushed or beaten, or seared (polenta) grain, for example, couscous grain.

Starch

In another aspect, the present invention relates to starch obtained from the grain of wheat plants described herein, wherein said starch has a high amylose content and a low amylopectin content. In a preferred embodiment of the invention, such starch is obtained from the grain of a wheat plant having a reduced level of SBEIIa protein, a reduced activity of the SBEIIa enzyme in the endosperm, or both compared to wild-type wheat. In another embodiment of the invention, the levels of both SBEIIa and SBEIIb activities are reduced, or the activity of all three enzymes SBEIIa, SBEIIb and SBEI are reduced compared to the activity of wild-type wheat.

In another aspect, the present invention relates to starch obtained from grain of wheat plants described herein, wherein the amylose content is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 90%. This starch is at least partially refined, that is, it is separated from at least one of the other components of the grain. Refined starch can be obtained from grain by grinding, for example wet grinding, which involves the separation of starch from protein, oil and fibers. The primary grinding product is a mixture or composition of starch granules, and therefore such granules containing the modified starch described herein are also included in the scope of the present invention.

Said starch may have an increased or decreased gelatinization temperature, and preferably, an increased gelatinization temperature. In specific embodiments of the invention, at least one of the temperatures corresponding to the beginning of the first peak, or the maximum of the first peak, increases by at least 3 ° C, at least 5 ° C, at least 7 ° With or at least 10 ° C, as measured by DSC, compared to the gelatinization temperature of starch extracted from wild-type wheat grain. In a specific embodiment of the present invention, the temperature increase is from 3 to 12 ° C. In a specific case, the gelatinization temperature may be lowered at the beginning of the first peak and increased at the top of the peak. In another embodiment of the invention, which does not exclude the previous embodiment, said starch may have a changed gelatinization temperature for the first peak, but have, basically, the same temperature for the second peak, which corresponds to the dissociation of amylose-lipid, as determined by DSC. In another embodiment of the invention, the starch has a reduced enthalpy during gelation, for example it detects a decrease in gelatinization temperature by at least 25% or at least 40% compared with the corresponding temperature for wild-type wheat starch.

In another embodiment of the invention, said starch contains an increased level of resistant starch, while its modified structure is characterized by specific physical properties. Such properties may include physical inaccessibility for hydrolyzing enzymes, which may be due to the altered morphology of starch granules; the presence of a valuable starch-associated lipid; altered crystallinity, altered distribution of amylopectin chain lengths, or any combination of these properties. A high amylose content also contributes to an increase in resistant starch levels.

The present invention also relates to starch obtained from a grain of a wheat plant described herein containing an increased amount of dietary fiber, preferably in combination with an increased level of resistant starch. This increase is also, at least in part, due to the relatively high level of amylose.

It is therefore apparent that the present invention relates to methods for producing wheat starch described in this application. In one embodiment of the invention, said method comprises the steps of producing wheat grain described herein and extracting starch from such grain. Wheat grain can be obtained by cultivating wheat plants described herein and collecting such grain, or it can be obtained from suppliers or importers of such grain.

Ways to reduce gene activity

The expression and / or activity of SBEIIa, SBEIIb, or other starch biosynthesis or modification genes can be altered by introducing one or more genetic modifications into a wheat plant. As used herein, the term “genetic modification” means any inherited change in the genome of a wheat plant that in a given environment affects the expression or activity of the gene of interest. Genetic modifications are mutations, such as point mutations, insertions, substitutions, inversions, duplications, translocations, and preferably deletions, and the insertion of one or more transgenes into the genome.

As used herein, the terms “nucleic acid molecule” and “nucleic acid sequence” mean a polymer consisting of nucleotides that can be single-stranded or double-stranded. This polymer may include DNA, such as, for example, genomic DNA or cDNA, RNA, mRNA, or any combination thereof. For introduction into wheat cells, the nucleic acid molecule can be chemically modified to improve delivery or increase stability, or it can be presented in protected form as part of a vector, such as a viral vector. A nucleic acid molecule can be obtained by cloning, or it can be synthesized by well-known methods. A nucleic acid molecule may comprise a coding chain, or a non-coding chain (antisense chain), or a combination thereof, such as, for example, is present in inverted repeat constructs. As for nucleic acid sequences that “match” a gene, the term “match” means the degree of similarity of the nucleotide sequences, that is, it means that the nucleotide sequence has a nucleotide sequence that is identical to the compared sequence of the gene or its specified part, or it has a nucleotide sequence that is completely complementary in terms of normal base pairing according to the Watson-Kr model An egg, either it is the RNA equivalent of such a sequence, for example mRNA, or it is cDNA derived from the mRNA of a gene.

The nucleotide sequences described herein are single-stranded nucleotides in the direction 5 ′ → 3 ′, which are indicated by standard single-letter abbreviations. The term “complementary” describes the similarity between two single-stranded nucleic acid molecules or sequences that hybridize by base pairing. So, for example, 5'-GAST-3'-pairs correspond to their complement 5'-AGTS-3 '. The terms “homology” or “homologous” mean the similarity or identity between two or more nucleotide sequences or between two or more polypeptide sequences in accordance with the context. The term “percent identity”, referring to nucleotide sequences, means the percentage of nucleotide matches between two nucleotide sequences aligned using a standardized algorithm, such as, for example, CLUSTAL V algorithm or Blastn or BLAST2 programs, which can be taken from the National Center for Biotechnological Information, and from the Internet at http://www.ncbi.nlm.nih.gov/BLAST/ , preferably using the default settings. Similarly, the term “percent identity” may refer to polypeptide sequences.

The term “gene”, including the SBEIIa and SBEIIb gene or other starch biosynthesis gene, or a gene encoding antisense molecules, co-suppressor molecules, ribozyme, duplex RNA molecules or the like, is used here in the broadest sense of the word and means classic a genomic gene having transcribed regions associated with regulatory regions, such as promoters, and transcription terminators, and polyadenylation sequences. Transcribed regions include transcribed, but not translated sequences (untranslated sequences, UTRs) and may include, but are not limited to, a protein coding region or introns that are spliced to form mature DNA, or any combination thereof. The term “gene” includes forms derived from cDNA corresponding to exons and RNA genes present in RNA genomes. The term “gene” is also used to describe synthesized or hybrid molecules encoding all or part of a functional product.

A “gene” present in a cell, and preferably in a wheat cell, directs the “expression” of a “biologically active molecule” or “gene product”, which may be an RNA or polypeptide. Such a process is usually transcription with RNA production and translation with protein production. The specified product can then be modified in a cell. RNA can be modified, for example, by polyadenylation, splicing, “capping”, “slicing” into fragments of 21-23 nucleotides, or exporting from the nucleus, or by covalent or non-covalent interaction with proteins. Proteins can be modified, for example, by phosphorylation, glycosylation or lipidation. All of these processes are covered by the term “gene expression" or other terms used here.

As used herein, the terms “ SBEIIa wheat gene” and “ SBEIIb wheat gene ” and related terms mean genes that have been identified in wheat as genes encoding SBEIIa or SBEIIb enzymes, respectively, and homologous genes present in other wheat varieties. These terms include, but are not limited to, the gene sequences listed in Table 1. It should be noted that, in the sequences of the SBEIIa and SBEIIb genes, there are natural modifications that are different for different wheat varieties. Homologous genes can be easily identified by one of ordinary skill in the art. The degree of sequence identity between homologous SBEIIa genes or proteins is at least 90%, the same applies to SBEIIb genes or proteins.

The genes used in the present invention can be obtained from the natural SBEIIa and SBEIIb genes or from other starch biosynthesis genes using standard recombinant DNA methods. As used herein, the term “recombinant nucleic acid molecule” or the like. means a sequence that does not occur in nature or which has a sequence created by artificially combining two or more other separate segments of the sequence. This artificial combination can be formed by chemical synthesis or, mainly, by artificially modifying individual segments of nucleic acids, for example, by genetic engineering methods well known in the art. The term “recombinant” includes nucleic acids that are modified only by the addition, replacement or deletion of a portion of the nucleic acid. In most cases, a recombinant nucleic acid may comprise a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector used, for example, to transform a cell.

In general terms, a gene can be mutagenized to produce one or multiple nucleotide substitutions, deletions and / or additions, such as, for example, codon modification. The nucleotide insertion derivatives of such genes are 5'- and 3'-terminal hybrids, as well as insertions of one or multiple nucleotides into the sequence. Variants of the insertion nucleotide sequence are variants in which one or more nucleotides are inserted into a predetermined site of the nucleotide sequence, although random insertion can also be detected by appropriate screening of the resulting product. Deletion variants are characterized by the absence of one or more nucleotides in a given sequence. Substitutional nucleotide variants are those in which at least one nucleotide in a given sequence has been deleted and another nucleotide has been inserted in its place. Such a substitution can be “silent,” that is, such a substitution does not lead to a substitution of the amino acid defined by this codon. Alternatively, conservative substitutions are carried out in order to replace one amino acid with another amino acid with a similar function. Typical substitutions are those carried out as follows.

Suitable residues for conservative amino acid substitutions:

Original balance Characteristic replacements Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn; Gln Ile Leu Val Leu Ile Val Lys Arg; Gln; Glu Met Leu Ile Phe Met; Leu Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile Leu

Transgenes

The expression and / or activity of the SBEIIa and SBEIIb genes or other genes responsible for the biosynthesis or modification of starch can be altered by introducing one or more transgenes into wheat plants. As used herein, the term “transgene” has a common meaning in the field of biotechnology and includes a gene sequence that has been produced or modified by recombinant DNA or RNA methods and which has been introduced into an organism or cell of interest, and preferably into a wheat cell. Such a transgene may include genetic sequences originating from an organism or cell, for example, an antisense sequence. Typically, a transgene includes an exogenous nucleic acid that does not originate from a specified organism or from a specified cell. The term “transgenic” refers to an organism or to a cell containing a transgene. The term “non-transgenic” means the absence of any transgene in the genome. The transgene is preferably integrated into the genome of an organism or cells to ensure stable inheritance.

Every person skilled in the art knows that expression of a gene or a complementary sequence in a cell requires that the gene is functionally linked to the promoter sequence. To achieve these goals, the promoter can be selected depending on the desired level of expression, and / or on the tissue, organ and type in which the expression should occur, and in particular, such endosperm-specific promoters are.

The introduction of a nucleic acid molecule under the control of the promoter regulatory sequence implies that the specified molecule must be in such a position that its expression is regulated by the promoter sequence. The promoter is usually, but not necessarily, located above or at the 5 ′ end of the nucleic acid molecule that it regulates. In addition, regulatory elements containing a promoter are typically located within 2 kb. from a gene transcription initiation site. In the design of heterologous promoter / structural gene combinations, it is generally preferred that the promoter is located at a certain distance from the gene transcription initiation site, which is approximately the same as the distance between the specified promoter and the gene that it regulates in nature (i.e., the gene from which this promoter is derived). As is known to those skilled in the art, some changes in this distance may be allowed if the promoter function does not change. Similarly, the preferred localization of an element of a regulatory sequence with respect to a heterologous gene placed under its control is determined by the localization of this element in nature (i.e., the gene from which it originates). And in this case, as is known to specialists, this distance can vary to some extent.

Examples of promoters suitable for use in the gene constructs of the present invention are promoters derived from genes of viruses, yeast, mold, bacteria, insects, birds, mammals and plants, and preferred promoters are promoters that are able to function in plant cells, and more preferably capable of being expressed in the endosperm of wheat. A promoter may regulate expression constitutively or inducibly, depending on the tissue in which expression occurs. Alternatively, expression may be differential, depending on the developmental stage at which expression occurs, or such expression may occur in response to an external stimulus, such as physiological stress or a change in temperature.

A method of reducing the activity of the SBEIIa gene or other starch biosynthesis gene may include the step of introducing the transgene into the regenerated wheat cell and regenerating the transgenic wheat plant from the transformed cell. The branching enzymes involved in the synthesis of amylopectin are SBEI, SBEIIa and SBEIIb, and the scope of the present invention encompasses a decrease in the expression level of only one SBEIIa gene or in combination with a change in expression levels of SBEIIb or SBEI genes . Therefore, the transgene (s) can (can) inactivate more than one of these genes. In addition, the inactivation of SBEIIb and / or SBEI can be direct, that is, the transgene (e.g., encoding duplex RNA, antisense RNA or ribozyme RNA, see below) is directly oriented to the expression of SBEIIb or SBEI genes , or it can mediate a change in the expression level SBEIIb or SBEI . So, for example, transgenic RNA can only be directed to the SBEIIa / RNA gene in terms of sequence identity or base pairing, but it can also lead to a decrease in SBEIIb or SBEI activity by changing the stability of the protein or its distribution in the endosperm. In another embodiment, the present invention includes a combination of altered SBEIIa activity and modification of one or more amylopectin synthesis enzymes, wherein said enzymes may be SSI, SSII, SSIII and branching enzymes such as isoamylase or pullulanase. The expression of any or all of these enzymes can be altered by introducing a transgene.

Several DNA sequences are known for amylopectin synthesis genes in wheat, and any of them can serve as the basis for the construction of transgenes in order to inactivate genes in wheat. Such genes are SBEIIa (GenBank, reg.No. Y11282, AF338431 and AF338432) and SBEIIb (WO 00/15810, WO 01/62934). The SBEI gene of wheat is described by Rahman et al. (1997) and Rahman et al. (1999). The SBEI sequence of Triticum tauschii , which is a highly homologous SBEI sequence of the wheat genome D, can be found in the description of published patent application WO 99/14314. Wheat SBEI gene cDNA sequence can be found in the GenBank database under registration number AF076679. Homologs of other amylopectin synthesis genes derived from barley or other closely related species can also be used to alter gene expression levels in wheat. Such genes or fragments thereof can be obtained by methods well known in the art, including PCR amplification or hybridization with labeled probes.

As used herein, the term “stringent hybridization conditions” means that hybridization can be carried out if the identity of the probe sequences and the target sequence is at least 90%, and preferably at least 95%. The following stringent hybridization conditions can serve as examples: incubation overnight in a solution containing 50% formamide, 5 × SSC (1 × SSC = 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6) , 5 × Denhardt's solution, 10% dextran sulfate and 20 μg / ml denatured fragmented carrier DNA, such as salmon sperm DNA, and subsequent washing of the hybridization carrier in 0.1 × SSC at approximately 65 ° C. Other hybridization and flushing conditions are well known to those skilled in the art and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), and in particular in Chapter 11.

The region (s) of homology used (s) to obtain the transgenic construct should be at least 85% identical to the region of the corresponding wheat gene, and preferably, it should be identical to the corresponding region at least 90%, and even more preferably 95-100%. It is also preferred that the transgene specifically targets amylopectin synthesis genes expressed in wheat endosperm and not having any effect, or having minimal effect on amylopectin synthesis in any other parts of the plant. This can be achieved using suitable regulatory sequences, such as endosperm specific promoters in the transgene.

Antisense sequences

Genetic engineering methods that allow you to change, and in particular, specifically reduce the activity of a gene in plants, such as wheat, are well known in the art. Such methods include the introduction of gene constructs for the expression of a suitable antisense molecule that is complementary to the RNA of the target gene and can hybridize with it. It is assumed that antisense molecules prevent translation or processing, or adversely affect the stability of the mRNA of the target gene, which leads to inactivation of gene expression. Methods for obtaining antisense sequences are well known in the art, and examples thereof can be found in US Pat. No. 5,190,131, European Patent Application 0467349-A1, European Patent Application 0223399-A1 and European Patent Application 0240208, which are incorporated herein by links. Application of methods using antisense sequences in plants is described by Bourque (1995) and Senior (1998). Bourque provides many examples of gene inactivation using antisense sequences in plant systems. This work also indicates that it is not necessary to achieve 100% inhibition of enzymatic activity, since it is obvious that partial inhibition will lead to significant changes in this system. Senior claimed that methods using antisense sequences are a very well-developed technique for modifying gene expression in plants.

Antisense molecules for the SBEIIa , SBEIIb , SBEI, or other biosynthesis or starch modification genes in wheat can be based on wheat mRNA sequences, or they can be derived from homologous DNA or mRNA sequences obtained from other species, such as barley. Antisense sequences may correspond to all transcripts or parts of the transcripts of any of these genes, or to sequences that control during their expression, for example, their splicing. Antisense sequences may correspond to the target coding region of the SBEIIa gene or another wheat gene, or the 5'-untranslated region (UTR) or 3'-UTR, or a combination thereof. The antisense sequence may be complementary to a portion of the intron sequences that can be excised during or after transcription, and preferably only to the exon sequences of the target gene. Given mainly the higher divergence of UTR, it can be said that targeting these regions gives a higher specificity for gene inhibition. In specific embodiments of the invention, the length of the antisense sequence is at least 19 adjacent nucleotides, at least 50, at least 100, at least 200, at least 500, or at least 1000 nucleotides, corresponding to the complement of the RNA sequence of the gene. In this case, a full-length sequence complementary to the transcript of the whole gene can be used. In a specific embodiment of the invention, the length of the antisense sequence is 100-2000 nucleotides. In other embodiments of the invention, the degree of identity of the antisense sequence to the complement of the target transcript is at least 85%, at least 90%, or 95-100%. Obviously, the antisense RNA molecule may contain unrelated sequences that can stabilize the molecule.

Co-suppression

Another method of molecular biology that can be used is co-suppression. The mechanism of co-suppression is not entirely clear, but it is obvious that it is involved in post-transcriptional inactivation (PTGS) of the gene and from this point of view it is very similar to many examples of antisense suppression. This mechanism ensures the introduction of an additional copy of the gene or its fragment into the plant in a sense orientation with respect to the promoter for its expression. The size of the semantic fragment, its correspondence to the regions of the target gene and the degree of identity of its sequence with the sequence of the target gene are the same as for the antisense sequences described above. In some cases, an additional copy of the gene sequence interferes with the expression of the target plant gene. Methods for implementing co-suppression are described in the patent application WO 97/20936 and in the description of Europatent 0465572.

Double-stranded RNA-mediated gene inactivation

Another method that can be used to introduce genetic modification into a wheat plant is gene inactivation mediated by duplex or double-stranded RNA. This method also provides for PTGS. In this method, DNA is introduced that regulates the synthesis, at least in part, of the double-stranded RNA product (s) homologous to the inactivated target gene. Therefore, this DNA includes both sense and antisense sequences, which, when transcribed into RNA, can hybridize to form a double-stranded RNA region. In a preferred embodiment of the invention, the sense and antisense sequences are separated by a spacer region containing an intron, which, when transcribed into RNA, is excised. It was shown that this arrangement of these sequences leads to an increase in the efficiency of gene inactivation. The specified double-stranded region may contain one or two RNA molecules transcribed from one or two DNA regions. The presence of a double-stranded molecule stimulates the response of the endogenous plant system, which leads to the destruction of both double-stranded RNA and the homologous RNA transcript in the target plant gene, and thereby to an effective decrease or elimination of the activity of the target gene. Implementation of these methods is described in Australian patent application 99/29514-A and in patent application WO 99/53050. In specific embodiments of the invention, the length of the hybridizable sense and antisense sequences is at least 19 adjacent nucleotides, at least 30, at least 50, at least 100, at least 200, at least 500 or at least 1000 nucleotides. In this case, a full-length sequence corresponding to the transcript of the whole gene can be used. In a specific embodiment of the invention, the lengths of these sequences are 100-2000 nucleotides. In other embodiments of the invention, the degree of identity of the sense and antisense sequences with the sequence of the target transcript is at least 85%, at least 90%, or 95-100%. Obviously, an RNA molecule may contain unrelated sequences that can stabilize a given molecule. The RNA molecule can be expressed under the control of the promoter of RNA polymerase II or RNA polymerase III. Examples of the latter promoter are tRNA or snRNA promoters. A double-stranded RNA molecule can also contain sequences of more than one gene attached to each other, and thereby affect many genes.

Ribozymes

The genetic modification responsible for the desired inactivation of gene expression in wheat may contain a nucleic acid molecule encoding one or more ribozymes. Ribozymes are RNA molecules that have an enzymatic or catalytic function that can cleave other RNA molecules at specific sites defined by one, and often two, hybridizing sequences. RNA cleavage inactivates the expression of the target gene. Ribozymes can also act as an antisense molecule that can provide for inactivation of a gene. Ribozymes contain one or more catalytic domains, and preferably hammerhead or hairpin domains located between hybridizing sequences. Other ribozyme motifs may be used, including RNase P, group I or II introns, and hepatitis delta virus motifs. Their description can be found in Europatent 0321201 and in US patent No. 6221661. The use of ribozymes for inactivation of genes in transgenic plants is demonstrated, for example, Wegener et al. (1994).

Genetic constructs / vectors

The present invention also relates to isolated nucleic acid molecules containing RNA or DNA, and preferably DNA, which encodes a gene inhibitory molecule. In some embodiments of the invention, the nucleic acid molecules encode an antisense, sense (co-suppressor), double-stranded RNA molecule or ribozyme molecule that target the wheat SBEIIa gene sequence and which inactivate its expression in the wheat grain endosperm. The present invention also relates to genetic constructs containing or encoding an isolated nucleic acid molecule containing one or more regulatory elements, such as promoters, enhancers, transcription termination sequences or polyadenylation sequences. Such elements are well known in the art. These gene constructs may also contain intron sequences that stimulate transgene expression in plants, and in particular in monocotyledonous plants, such as wheat. The term “intron” is used here in its usual meaning and means a gene segment that is transcribed, but does not encode a protein, and which is excised from RNA before translation. Introns can be introduced into the 5'-UTR or into the coding region if the transgene encodes the translated product, or into any other region of the transcribed region, if it does not encode this product.

In addition, the present invention relates to vectors, for example, plasmid vectors containing gene constructs. The term “vector” includes an expression vector capable of in vivo or in vitro expression, and a transforming vector that can be transmitted from one cell or organism to another cell or organism. These vectors contain sequences that allow their replication in cells, for example, in prokaryotic cells, such as E. coli or Agrobacterium . In a specific embodiment of the invention, said vector is a binary vector comprising a T-DNA sequence defined by at least one T-DNA boundary sequence that can be introduced into wheat cells. The present invention also relates to cells containing vectors, for example Agrobacterium, or to wheat cells, which may be regenerable cells, such as shield cells of an immature embryo. Alternatively, these cells may be transformed wheat cells containing a transgene.

Promoters / Terminators

In another embodiment of the invention, the transgenic or other genetic construct of the present invention includes a transcription initiation region (promoter) that can provide regulated or constitutive expression in the wheat endosperm. The promoter may be selected from an endosperm-specific promoter (such as a high molecular weight glutenin promoter, a wheat SSI promoter, a wheat SBEII promoter, a wheat GBSS promoter) or promoters that are not specific for the endosperm (such as a ubiquitin promoter or CaMV35S promoter or a reinforced 35S promoter ) This promoter can be modulated by factors such as temperature, light emission, or stress. Typically, the promoter should be located on the 5'-end of the expressed gene sequence. This construct may also contain other elements that enhance transcription, such as nos-3′- or os-3′-polyadenylation regions or transcription terminators. These DNA regions are introduced into vectors containing the corresponding sequences of the selective marker gene or other elements, or into vectors that undergo co-transformation with vectors containing these sequences.

Wheat Transformation Methods

Methods of transformation of monocotyledonous plants, such as wheat, which are used to introduce genetic modification into a plant by embedding exogenous nucleic acid and to regenerate plants from protoplasts or immature plant embryos, are well known in the art, see, for example, Becker et al., 1994, Cheng et al., 1997, He et al., 1994, Hess et al., 1990, Nehra et al., 1994, Vasil et al., 1992, Vasil et al., 1993, Weeks et al., 1993, Weir et al., 2001, Australian patent application No. 75460/94, European patent application No. 709462, publication of international patent application No. WO 93/04178, WO 89/12012, WO 94/13822 and WO 99 / 14314. Vectors carrying the desired nucleotide sequence or genetic construct and a selectable marker can be introduced into the regenerated tissue cells of cultivated plants or wheat explants, or into suitable plant systems such as protoplasts. The selectable marker gene can provide resistance of wheat cells to antibiotics or herbicides, or it can provide utilization of substrates, such as mannose. A selective marker preferentially confers resistance to asulam, geneticin or hygromycin on wheat cells. Regenerated wheat cells mainly come from the shield of immature embryos, mature embryos, callus originating from these embryos, or from the meristem tissue.

The transformed plant may contain a selective marker gene, or such a gene can be removed during or after regeneration, for example, by excising a selective marker gene from the genome or by segregating a selective marker gene from an SBEIIa- inhibiting transgene.

Plants where the transgene or mutation is integrated into the chromosome can be screened for the indicated transgene, for example, using a suitable nucleic acid probe specific for the transgene or phenotype. To determine the presence of a transformed plant, any of the available methods can be applied. So, for example, to amplify sequences that may be unique to a transformed plant, a polymerase chain reaction (PCR) can be carried out with the detection of amplified products using gel electrophoresis or other methods. DNA can be extracted from plants by standard methods and by PCR using primers that will recognize transformed and non-transformed plants. For example, primers can be designed that amplify the DNA region of the transforming vector into the construct, and a reverse primer constructed from the gene of interest. These primers will amplify the fragment only if this plant is successfully transformed. An alternative method for confirming a positive transform is Southern blot hybridization, well known in the art. Plants that are transformed or mutant can also be identified, that is, differentiated from untransformed plants or wild-type plants by their phenotype, for example, determined by the presence of a selective marker gene, or by the presence of a particular protein by immunological methods, or by the absence of protein, for example , by the absence of SBEIIa protein in the endosperm detected by ELISA or Western blot analysis. The indicators used for screening such plants can be phenotypic signs of grain, determined, for example, by visual observation or an assessment of the wrinkling of the grain, or by analysis for an increased content of amylose or microscopic analysis for the presence of birefringence.

Mutation

The introduction of genetic modification, which leads to a decrease in the activity of the enzyme SBEIIa or another starch biosynthesis enzyme in the wheat endosperm, can also be achieved by introducing suitable mutations into the corresponding gene or into the regulatory sequences of the gene. In the context of the present invention, the term “induced mutation” means an artificially induced genetic modification that may result from chemical mutagenesis or mutagenesis induced by radiation or biological mechanisms, for example, the insertion of a transposon or T-DNA. The level of gene inhibition can to some extent determine the properties of the resulting starch. Mutations can be truncation or silent mutations; it is known that these mutations have a significant effect on the nature of starch, however, the structure of the modified starch can also be the result of a facial (spreading) mutation, which sufficiently reduces the activity of the enzyme responsible for the synthesis of amylopectin, and allows starch or wheat grain with the desired properties. Other chromosomal rearrangements may also be effective, and insertions, deletions, inversions, duplications, or point mutations can be such chromosomal rearrangements. As used herein, the term “null mutation” (or “silent mutation”) means a mutation that results in a complete or almost complete loss of activity of the gene of interest, for example, to the extent that gene activity is no longer detected.

The SBEIIa gene is localized on the long arm of chromosome 2. Moreover, it is preferable that mutations in this gene or in other genes, in particular deletion mutations, be localized in the desired gene, for example, in the SBEIIa gene, or that they extend to the SBEIIb gene linked to it in case of double mutation. In this case, the gene includes the promoter region and transcription / polyadenylation termination signals, as well as the transcribed region. The transcribed region includes the protein-coding (s) region (s) and 5'-untranslated and 3'-untranslated regions of mRNA, as well as any intron regions that may be present in this region. Mutations in a gene can be present in any region or in a combination of regions of a gene and can arise as a result of modification of only one nucleotide, for example, as a result of mutations with a shift of the reading frame in the coding region, or deletion of the entire gene. Preferred are plants that are homozygous for the indicated genetic modification.

The size of deletions can be limited to about one or several hundred, and possibly 500 thousand pairs of nucleotides. In some embodiments of the invention, such a deletion encompasses less than several thousand nucleotide pairs or less than 5 thousand nucleotides. Although the present invention may include larger deletions, covering most of the long arm of chromosome 2 of the corresponding genome, however, these deletions are not preferred, since the long arm of chromosome 2 has a number of other genes located in it, affecting the power of the wheat plant. Thus, since large deletions have a negative effect on the plant's power, and therefore on its commercial value, it is desirable that at least a large part of the long arm of chromosome 2 is retained. In a preferred embodiment, a large part of the arm of chromosome 2A is retained.

Mutagenesis can be achieved by exposure to a chemical or radiation, for example, by treating the seeds with EMS or sodium azide (Zwar & Chandler, 1995) or gamma radiation. Isolation of mutants can be carried out by screening plants or seeds subjected to mutagenesis. For example, a mutagenized population of wheat can be screened for a high amylose content in the grain and / or for the distribution of longer amylopectin chains exceeding the normal length, or for the absence of SBEIIa protein evaluated by ELISA, or for altered grain morphology (Green et al., 1997). Preferably, screening is carried out for a wheat genotype that already does not contain one of the SBE activities, for example, for an SBEIIb negative phenotype. These mutations can then be introduced into plants with the desired genetic background by crossing a mutant plant with a plant having the desired genetic background, and performing a suitable number of backcrossing procedures to eliminate the undesirable parent phenotype.

In another embodiment of the invention, the mutation affects the expression or activity of both SBEIIa and SBEIIb genes in wheat. The identification of this mutation was due to the unexpected discovery of the fact that these two genes are closely linked in wheat, unlike corn or rice. Deletions in one gene can easily spread to another gene with the formation of a silent allele (silent mutation) for both genes. These data also facilitate screening of natural variants that are mutant in both genes in at least one wheat genome, and facilitate screening for wheat production with combined mutations in both genes in two or three genomes. Such wheat has a high amylose content and is a non-transgenic source for producing wheat grain and products from this grain.

Mutations in the genes encoding the SBEIIa enzyme or other enzymes involved in the synthesis of amylopectin will mainly provide an increased content of amylose. The amount of amylose per grain can be increased as a result of a decrease in the flow of carbon from amylopectin and its transfer to amylose, or it can be reduced if there is a significant decrease in the production of starch for wheat grain. In any case, the relative level of amylose increases as the percentage of starch content increases.

It was reported that barley seeds were obtained with starch granules having a deformed shape and high amylose content (Morell et al., 2003), and corn seeds with starch having a low amylopectin content (LAPS) and approximately 90% amylose (Sidebottom et al. , 1998).

“Birefringence” means the ability of a substance to refract a ray of light in two directions, and such refraction can be observed under a polarizing microscope in the form of a “dark” cross called the “Maltese cross” on each starch granule. Birefringence is an indicator of the degree of ordered structural organization of polymers within these starch granules (Thomas & Atwell, 1999). The loss of the ability of starch granules to birefringence, generally speaking, correlates well with an increased content of amylose.

Wheat for food

In another aspect, the present invention relates to wheat, which can be used to produce food products and whose grain contains starch having a high relative amylose content and a low amylopectin content. Moreover, it is preferable that the wheat plant from which the grain was obtained had a reduced level of SBEIIa activity in the endosperm during its development. The wheat plant of the present invention can be used to make food products. The manufacture of such food products may include the production of flour, dough or other products that can serve as ingredients for the manufacture of food products.

When choosing the desired genetic background of such wheat should take into account yield and other characteristics. If it is necessary to breed winter or spring wheat species, then such characteristics can be agronomic productivity, disease resistance and resistance to abiotic stress. In Australia, it may be desirable to cross-wheat with phenotypic traits of modified starch with wheat varieties such as Baxter, Kennedy, Janz, Frame, Rosella, Cadoux, Diamondbird or other known varieties. The above varieties are suitable for cultivation in specific regions of Australia, and other varieties are suitable for other agricultural regions. In this case, it is preferable that the wheat variety of the present invention yield at least 80%, more preferably at least 90%, and even more preferably at least 95%, compared with the harvest of wild-type varieties, at least in some agricultural regions. This crop can be easily measured in controlled field trials.

In other embodiments of the invention, the starch content of the grain is at least about 25%, 35%, 45%, or 55-65% (w / w). In industrial cultivation, wild-type wheat has a starch content of about 55-65%, which partly depends on the cultivated variety. Alternatively, the grain of the present invention has a starch content of at least 90% with respect to the grain of equivalent but unmodified wheat. The lower starch content compared to the starch content in the wild-type plant is due to low levels of amylopectin. Even with a lower starch content, such grain can be used for the manufacture of industrial food products, because these products will have a relatively high value, due to the high content of amylose. Other desirable characteristics are the milling properties of the grain, and in particular the hardness of the grain. Another factor that can add value to a wheat plant is a higher degree of starch extraction from the grain, and the higher the extraction rate, the more valuable the grain. Another sign that can affect the commercial value of the plant is the shape of the grain, since the ease of grinding of the grain or other parameters depends on it. For example, elongated grain is more difficult to grind and process.

Poured grain may be preferable from the point of view of achieving higher yields, and in this case some useful effects of the present invention can be achieved, such as producing starch with higher levels of amylose, or, alternatively, starch with a modified distribution of its chain lengths. Thus, it is preferable that the grain has a phenotype of non-wrinkled grain. However, in other aspects of the present invention, grain with a lower bulk can be obtained. So, for example, in a less poured grain, the content of the aleuron layer, germ or protein with respect to starch in a less poured grain may be higher, which makes it possible to obtain wheat flour or other product with a high content of valuable components, such as an aleurone layer or protein. Thus, a product with a high aleurone layer can be enriched with certain vitamins, such as folate, or it can be enriched with minerals such as calcium, and in combination with elevated levels of resistant starch, a synergistic effect, such as increased absorption of minerals by thick intestines.

Starch can be easily isolated from wheat grains using standard methods, for example, the method of Schulman et al., (1991). In large-scale production, wet or dry grinding may be used. The size of starch granules is an important factor in the industrial processing of starch where larger granules A are separated from smaller granules B. Starch obtained from the grain of a wheat plant of the present invention has a high relative amylose content.

Physical characteristics of modified starch

Gelatinization is a thermally regulated collapse (disruption) of the molecular structure in starch granules with an excess of water, with simultaneous and irreversible changes in their properties, such as grain swelling, crystallite melting, loss of birefringence, increase in viscosity and solubilization of starch. High amylose starch derived from ae mutants ( an amylose chain extender) of maize showed a higher gelation temperature than normal maize starch (Fuwa et al., 1999, Krueger et al., 1987). On the other hand, starch derived from sex6 mutants that lack starch synthase II activity has lower gelatinization temperatures, and the enthalpy for peak gelatinization is lower than the enthalpy for control plants (Morell et al., 2003) .

In another aspect of the present invention, starch has an altered gelation temperature as measured by differential scanning calorimetry. This temperature may be higher or lower than the gelatinization temperature of starch of wild-type plants. The altered gelation temperature may be combined with a relatively high amylose content. The starch gelatinization temperature of wild-type wheat plants is typically about 61 ° C (Rahman et al., 2000) for the temperature of the first peak, defined as the peak onset temperature and measured by differential scanning calorimetry.

Starch can also be characterized by the degree of swelling in excess hot water compared to wild-type starch. The volume of swelling is usually measured by mixing starch or flour with excess water and heating to high temperatures, usually more than 90 ° C. The sample is then collected by centrifugation, and the swelling volume is expressed as the mass of precipitated material divided by the dry mass of the sample. A low level of swelling is desirable if an increase in starch content in the food product, and in particular in the hydrated food product, is necessary.

The starch structure in wheat of the selected forms in accordance with the present invention may also differ in that it has a lower crystallinity than normal starch isolated from wheat. The lower crystallinity of starch is obviously also associated with an increase in organoleptic properties and gives the starch a softer consistency when chewed. Thus, starch may also have a lower degree of crystallinity, due to a decrease in the activity levels of one or more amylopectin synthesis enzymes. Crystallinity is usually investigated by x-ray crystallography.

One parameter of the modified structure of amylopectin is the distribution of chain lengths or the degree of polymerization of starch. The distribution of chain lengths can be determined by electrophoresis in a carbohydrate in the presence of fluorophore (FACE) after isoamylase branching. The starch amylopectin of the present invention may have a chain length distribution in the range of 5 to 60, which is greater than the values obtained for the distribution of starch of wild-type plants after branching. Starch with a longer chain will also detect a corresponding decrease in branching frequency. Thus, the distribution of chain lengths of aminopectin in a given starch may also show a tendency to increase the number of longer chains in the starch.

Food properties

Starch is the main source of carbohydrates in human food, and the grain of the present invention and products derived from it can be used to prepare food products. Such a food product may be consumed by humans or animals for food, for example, in the form of feed used in animal husbandry, or pet food. The grain obtained from the modified wheat plant can be successfully used in food technology, and therefore the present invention includes ground, crushed, crushed, crushed or rolled grain or products obtained from processed or whole grain of the above wheat plants, including flour . These products can then be used to make various food products, for example flour products, such as bread, cookies, biscuits, etc., or food additives, such as thickeners or binders, or to make drinks, noodles, pasta or instant soups. The grain or the products obtained therefrom in accordance with the present invention are suitable for making breakfast cereals or for use as pressed products. The high amylose starches in accordance with the present invention can also be used to produce highly gelling gels that can be used in the confectionery industry, or when it is necessary to reduce the melting and curing times. They can also be used as coatings, for example, to reduce the absorption of oil by deep-fried potatoes or other food products.

Alimentary fiber

In the present description, the term “dietary fiber” means a carbohydrate and carbohydrate breakdown products that are not absorbed in the small intestine of a healthy person, but enter the large intestine. Such products include resistant starch, β-glucan and other soluble and insoluble carbohydrate polymers. It is assumed that part of the fermentable carbohydrates is utilized, at least in part, in the colon by the microflora present there.

The starch of the present invention preferably contains relatively high levels of dietary fiber, and more preferably, amylose. In accordance with the present invention, the content of dietary fiber in the grain may or may not depend only on the increase in the relative content of amylose in the endosperm.

In some aspects of the invention, a combination of an aleurone layer and an embryo in combination with high levels of dietary fiber is contemplated. In particular, this may apply to cases where there are elevated relative levels of an aleuron or embryo in the grain. If the wheat grain is slightly wrinkled, this means that the endosperm is present in small amounts, and the aleuron layer and the germ are present in relatively high quantities. Thus, such wheat has a relatively high level of certain valuable elements or vitamins in combination with an increased level of resistant starch, where such elements are divalent cations, bioavailable Ca ⁺⁺ , and vitamins are folate or antioxidants such as tocopherols or tocotrienols. One of the specific forms of the grinding product may be a ground product in which an aleurone layer is present. To increase the amount of the aleurone layer in the ground product, a specific grinding technique can be applied. Thus, any product obtained from ground grain or grain processed in any other way, so that it includes the aleurone layer and the germ, will contain additional nutrients, which will avoid the need to add these elements from separate sources.

Resistant starch

The term “resistant starch” is defined as starch and starch hydrolysis products that are not absorbed in the small intestine of a healthy person, but enter the large intestine. Thus, resistant starch does not include products hydrolyzed and absorbed in the small intestine. Resistant starches include physically inaccessible starch (RS1 form), resistant starch granules (RS2), retrograde starch (RS3), and chemically modified starch (RS4). In accordance with the present invention, the modified structure of starch, and in particular starch with a high amylose content, leads to an increase in the level of resistant starch used in food. This starch may have an RS1 form, which is somewhat inaccessible for enzymatic hydrolysis. The association of starch and lipid, assessed by the crystallinity of the V complex, also increases the level of resistant starch.

It should be noted that the advantage of the present invention is that it allows to obtain products containing specific beneficial nutrients, and, in addition, does not require the mandatory modification of starch or other components of wheat grain. However, such a modification of starch or other components of the grain may be desirable, and the present invention also includes such modified components. Modification methods are well known to those skilled in the art and provide for the extraction of starch or other components by standard methods and the modification of starch to increase levels of its resistant forms. Starch can be modified by heat treatment and / or wet processing, physical processing (e.g. grinding in a ball mill), enzymatic processing (using, for example, α- or β-amylase, pullulanase, etc.), chemical hydrolysis (wet or dry using liquid or gaseous reagents), oxidation, crosslinking with bifunctional reagents (for example, sodium trimetaphosphate, phosphorus oxychloride) or carboxymethylation.

Glycemic index

The glycemic index (GI) means the rate of hydrolysis of starchy foods and is determined by comparing the effects of the test food and the effects of foods such as white bread or glucose on the increase in blood glucose concentration. The glycemic index is an indicator of the possible effect of a food product on serum glucose concentration after a meal and the need for insulin to ensure blood glucose homeostasis. One of the important characteristics of the food products of the present invention is the low glycemic index value. In addition, these foods may have a low level of final hydrolysis, and therefore a relatively low calorie content. A low-calorie product can be obtained from flour produced from ground wheat grains. Such foods can give a feeling of satiety, improve bowel function, reduce the concentration of glucose and lipids in serum after eating, and can also serve as the basis for the manufacture of other low-calorie foods.

Application in the production of non-food products

The present invention relates to modified or improved starches having elevated levels of amylose or decreased levels of amylopectin, that is, starches whose properties meet any of various industrial production requirements. Starches are widely used in the manufacture of non-food products, including the manufacture of films, paper, textiles, corrugated products and in the production of adhesives (Young, 1984), for example, as dressing agents. Wheat starch can be used as a substrate in the production of glucose syrups or in the production of ethanol. In some starch applications, the use of unmodified starch is limited by its physical properties and often requires chemical modification, which is an expensive procedure or has other disadvantages. The present invention relates to starch, which does not require modification after its extraction from grain, which, in particular, is due to the reduced content of amylopectin in combination with other physical properties. For example, properties such as pour point, shear resistance, film strength and / or water resistance of starches and products made from grain in accordance with the present invention can be changed. The resulting starch can also be used for the manufacture of biodegradable cushioning bulk packaging material, which can serve as a replacement for polystyrene or other packaging materials.

It should be noted that although its various features are described in aspects of the present invention, however, the present invention may include a combination of two or more aspects thereof.

Examples

Example 1. Materials and methods

Determination and analysis of carbohydrate levels

Starch was isolated from wheat grain by the method of Schulman et al. (1991). Starch content was determined using a total starch assay kit supplied by Megazyme (Bray, Co Wicklow, Republic of Ireland). Then the content of this starch was compared with the starch content in the control plants. After subtracting the mass of starch from the total mass of grain, the total amount of grain without starch was determined, which made it possible to determine whether a decrease in the total mass of grain was associated with a decrease in starch content.

The amylose content in starch samples was determined by the calorimetric (iodometric) method of Morrison & Laignelet (1983), which was slightly modified as described below. Approximately 2 mg of starch was accurately weighed (to the nearest 0.1 mg) in a 2 ml screw cap tube equipped with a rubber pad. To remove lipid, 1 ml of 85% (v / v) methanol was mixed with starch, and the tube was heated at 65 ° C in a water bath for 1 hour, stirring occasionally. After centrifugation at 13000 × g for 5 minutes, the supernatant was carefully removed and the extraction steps were repeated. The starch was then dried at 65 ° C for 1 hour and dissolved in a solution of urea-dimethyl sulfoxide (UDMSO; 9 volumes of dimethyl sulfoxide per 1 volume of 6M urea) using 1 ml of UDMSO per 2 mg of starch (weighed as described above). The resulting mixture was immediately intensively mixed and incubated at 95 ° C in a water bath for 1 hour with periodic stirring to completely dissolve the starch. An aliquot of the starch-UDMSO solution (50 μl) was treated with 20 μl of I ₂ -KI reagent containing 2 mg of iodine and 20 mg of potassium iodide per 1 ml of water. The mixture was adjusted to 1 ml by adding water. The optical density of the mixture at 650 nm was measured by transferring 200 μl of the mixture into a microplate and reading the optical density on an Emax Precision Microplate Reader (Molecular Devices, USA). Standard samples containing from 0 to 100% amylose and from 100% to 0% amylopectin were obtained from potato amylose and corn (or potato) amylopectin (Sigma) and processed in the same way as for the test samples. Amylose content (percentage of amylose) was determined by the optical density using the regression equation derived from the optical density of standard samples. An analysis of the amylose / amylopectin ratio of unbranched starch can be carried out as described by Case et al. (1998), or the HPLC method for the separation of branched starches described by Batey & Curtin (1996).

The distribution of chain lengths in starch can be analyzed by fluorophore carbohydrate electrophoresis (FACE) using a capillary electrophoresis device as described by Morell et al. (1998) after the depletion of starch samples. The gelatinization temperature profiles of starch samples can be determined using Pyris 1 differential scanning calorimetry (Perkin-Elmer, Norwalk, CT, USA). The viscosity of starch solutions can be measured using a high-speed viscosity analyzer (RVA, Newport Scientific Pty Ltd, Warriewood, Sydney), for example, under the conditions described by Batey et al., 1997. The measured parameters may be maximum viscosity (maximum viscosity of hot paste), stability during storage, final viscosity and paste formation temperature. The swelling volume of flour or starch can be determined by the method of Konik-Rose et al. (2001). Water absorption was measured by weighing the sample before and after mixing the flour or starch sample in water at a certain temperature and after collecting the gelled material.

Β-glucan levels can be determined using a kit supplied by Megazyme (Bray, Co Wicklow, Republic of Ireland).

Analysis of protein expression in the endosperm

The specific expression of the protein in the endosperm was analyzed using Western blot analyzes. The endosperm was excised from all maternal tissues and approximately 0.2 mg samples were homogenized in 600 μl of 50 mM KPi buffer (42 mM K ₂ NRA ₄ and 8 mM KH ₂ PO ₄ ), pH 7.5, containing 5 mM EDTA, 20% glycerol 5 mM DTT and 1 mM Pefabloc. The ground samples were centrifuged at 13000 × g for 10 minutes, and then aliquots of the supernatant were taken and frozen at -80 ° C until use. To estimate the total protein content, a BSA standard curve was constructed using 0, 20, 40, 60, 80, and 100 μl aliquots of 0.25 mg / ml BSA standard. Samples (3 μl) were brought to a volume of 100 μl by adding distilled water, and 1 ml of Coomassie + protein reagent was added to each of these samples. After 5 minutes, based on the standard curve, the absorbance was read at 595 nm using a sample without BSA (0 μl aliquots) as a control, and protein levels in the samples were determined. Samples containing 20 μg of total protein from each endosperm were subjected to electrophoresis in an 8% non-denaturing polyacrylamide gel containing 0.34 M Tris-HCl (pH 8.8), acrylamide (8.0%), ammonium persulfate (0.06 %) and TEMED (0.1%). After electrophoresis, proteins were transferred onto a nitrocellulose membrane as described by Morell et al. (1997), and subjected to an immune reaction with antibodies against SBEIIa or SBEIIb.

Example 2. Genetic constructs for modifying the expression of SBEIIa and SBEIIb of wheat

To reduce the expression level of SBEIIa or SBEIIb genes in wheat, duplex RNA (dsRNA) constructs were created. In such designs, the desired nucleic acid sequence corresponding to part of the genes SBEIIa or SBEIIb, are both in the sense and in the antisense orientation relative to the promoter, whereby the expressed RNA comprised complementary regions that were capable of base-pairing and form a duplex or double stranded RNA. The spacer region between the sense and antisense sequences contained an intron sequence, which, when transcribed as part of RNA in a transformed plant, should be spliced to form a rigid “hairpin” duplex structure. It has been found that incorporation of an intron increases the efficiency of gene inactivation reported by duplex RNA constructs (Smith et al., 2000). The desired nucleic acid was attached to the promoter sequence of high molecular weight glutenin (HMWG) (promoter of the Dx5 subunit gene, Reg.No. X12928, Anderson et al., 1989) and to the sequence terminator of the Agrobacterium nopaline synthase gene (nos 3 '). This construct provides endosperm-specific expression of dsRNA sequences.

The duplex RNA construct for SBEIIa contained a 1536 bp nucleotide sequence amplified by PCR from wheat SBEIIa gene (GenBank, Reg. No. AF338431, see FIG. 1). This construct included a 468 bp sequence containing whole exons 1 and 2 and a portion of exon 3 (at nucleotide positions 1058-1336, 1664-1761 and 2038-2219 in FIG. 1) with restriction Eco RI- and Kpn I- sites on both sides (fragment 1); 512 bp sequence consisting of parts of exons 3 and 4 and the whole intron 3 of the SBEIIa gene (at nucleotide positions 2220-2731 in FIG. 1) with Kpn I and Sac I sites on both sides (fragment 2); and 528 bp fragment consisting of full-sized exons 1, 2 and 3 of the SBEIIa gene (at nucleotide positions 1058-1336, 1664-1761 and 2038-2279 in Fig. 1) with Bam HI and Sac I sites at to both sides (fragment 3). Then fragments 1, 2 and 3 were ligated so that the sequence of fragment 3 was attached to fragment 2 in the antisense orientation with respect to fragment 1. First, duplex RNA constructs were generated in the pDVO3000 vector, which contained the HMWG and nos-3'- promoter sequence Terminator. The gene construct in the pDVO3000 vector was designated pDVO3-IIa, and the gene construct of the duplex RNA was designated ds-SBEIIa.

The strategy for constructing SBEIIa duplex RNA was similar. The SBEIIb construct contained a fragment containing 1607 bp amplified by PCR from the SBEIIb gene of wheat (the sequence is shown in FIG. 2). This design included a 471 bp sequence containing whole exons 1 and 2 and a portion of exon 3 (at nucleotides 489-640, 789-934 and 1598-1769 in FIG. 2) with restriction Eco RI- and Kpn I- sites on both sides (fragment 1), 589 bp sequence consisting of parts of exons 3 and 4 and the whole intron 3 of the SBEIIb gene (at nucleotide positions 1770-2364 in figure 2) with Kpn I and Sac I sites on both sides (fragment 2) and 528 bp fragment consisting of full-sized exons 1, 2 and 3 of the SBEIIb gene (at the nucleotide positions 489-640, 789-934 and 1598-1827 in figure 2) with Bam HI - and Sac I-sites on either side (Fragment 3). Then fragments 1, 2 and 3 were ligated so that the sequence of fragment 3 was attached to fragment 2 in the antisense orientation with respect to fragment 1. The gene construction of duplex RNA SBEIIb in the vector pDVO3000 was designated pDVO3-IIb, and the gene construction of duplex RNA was designated DC-SBEIIb. These structures are shown schematically in FIG. 3.

Then, each of the ds-RNA expression clusters was cut with the restriction enzyme Xho I and inserted into the pGB53 and pBIOS340 binary transforming vectors. pGB53 was constructed from pSB11 (Komari et al., 1996) by inserting a gene encoding asulam resistance (sul) and regulated by the rice actin promoter to form a unique Xho I site adjacent to the right end of T-DNA to introduce the gene of interest. Similarly, pBIOS340 was constructed from pSB1 (Komari et al., 1996) by introducing an nptII gene encoding resistance to kanamycin and geneticin and regulated by the rice actin promoter to form a unique Xho I site adjacent to the right end. The SBEIIa constructs in pGB53 and pBIOS340 were designated pCL51 and pCL59, respectively, and the SBEIIb constructs in pGB53 and pBIOS340 were designated pCL54 and pCL60, respectively.

Example 3: Wheat Transformation

Gene constructs for transformation of wheat were introduced by electroporation into the inactivated strain LBA4404 Agrobacterium tumefaciens , carrying the plasmid vir pAL4404 and pSB1 with its subsequent selection on a medium with spectinomycin. Transformed Agrobacterium strains were incubated in cured YEP medium at 27 ° C for 2 days. Then the bacteria were collected and resuspended in TSIM1 medium (MS medium with 100 mg / L myoinositol, 10 g / L glucose, 50 mg / L MES buffer, pH 5.5) containing 400 mM acetosyringone to an optical density of 2.4 at 650 at nm for inoculation of wheat.

Wheat plants (grade HB1, a spring wheat variety obtained from Nickerson Seeds Ltd. Rothwell, Lincs.) Were grown in a greenhouse in a day-night mode at 22/15 ° C. with daylight for 16 hours. Shoots were harvested about 14 days after flowering (embryos were about 1 mm long) when the wheat shoot reached 50 cm. All leaves from the shoots were removed except for the flag leaf, which was washed to remove contaminating fungal spores. Then the scales of each spikelet and the lower floral scales of the first two flowers were carefully removed to expose the immature seed. Generally speaking, only two of these seeds in each spikelet have no shell. This procedure was carried out along the entire length of the inflorescence. Then the ears were treated by spraying 70% IMS to quickly sterilize the surface.

Immature seeds were inoculated with Agrobacterium suspensions (1 μl) using a 10 μl Hamilton syringe at approximately the flap junction: endosperm so that all exposed seeds were inoculated. Then the shoots were placed in water, covered with a transparent plastic bag to prevent seed dehydration and placed in an illuminated incubator for 3 days at 23 ° C, with 16-hour daylight and illumination of 45 E · m ^-2 · s ^-1 PAR. After a 3-day co-cultivation, the inoculated immature seeds were removed and the surface was sterilized with 70% ethanol (30 sec), and then treated with 20% bleach (Domestos, 20 min), after which they were thoroughly washed in sterile distilled water. Immature embryos were isolated under aseptic conditions and placed upside down (20 embryos per plate) in W3T medium (MS medium, to which 20 g / l sucrose and 2 mg / l 2,4-D were added and 6 g / l were cured) agarose type I, Sigma) with the addition of 150 mg / l of timentin (W3T). The cultures were kept at 25 ° С and under illumination (at 16-hour daylight and illumination of 80 E · m ^-2 · s ^-2 PAR). Embryo scar development was evaluated 5 days after isolation, and this scar was removed if necessary to accelerate callus production. The embryos were kept on W3T medium for 4 weeks, and then 2 weeks after isolation they were transferred to fresh medium and evaluated for embryogenesis.

After culturing for 4 weeks, the callus originating from the inoculated embryos was similar to the control callus obtained from uninoculated embryos grown on W3T medium. Obviously, the presence of bacteria did not lead to a significant decrease in callus embryogenesis obtained from inoculated embryos. Embryogenic callus was transferred to W3 medium containing 2 mg / L asulam (if the pGB53 derivative was used) or 25 mg / L Geneticin (pBIOS340 derivatives) and 150 mg / L Timentin (W32AT). The calli were kept on this medium for another 2 weeks, and then each callus was cut into 2 mm pieces and again plated on W32AT medium. Control embryos obtained from inoculums with LVA4404 that did not contain binary vector constructs did not produce transformed callus on selective media.

After cultivation for another 2 weeks, the entire tissue was evaluated for the development of embryogenic callus: each callus, showing signs of continuous development 4 weeks after its placement on selective medium, was transferred to a regeneration medium (in an RMT-MS medium with 40 g / l maltose and 150 mg / l timentin, pH 5.8, cured 6 g / l agarose type I, Sigma). Shoots were regenerated on this medium for 4 weeks, and then they were transferred to MS30 medium containing 150 mg / l of timentin, to stimulate shoot growth and to root them. Then the young plants were transferred to the soil mixture and kept on a rack with aerosol irrigation for two weeks and, finally, transferred to the greenhouse.

This method processed a total of 3217 embryos using pCL54 or pCL60 (dc-SBEIIb) and 2010 embryos using pCL51 or pCL59 (dc-SBEIIa), after which 61 plants were regenerated from calluses for IIb transformation, and for IIa transformation 31 plants were regenerated. Survival on selective medium suggests that these plants were successfully transformed with a gene construct. It is assumed that the vast majority, but not all, of plants that were transformed with a selective marker gene had an SBEIIa- or SBEIIb-inhibitory gene integrated in their genome, and these plants can be easily identified, as described in the following examples.

The selection of many stable integration transformants with good ability to regenerate obtained in these experiments showed that the method of seed transformation by inoculation used here is as effective as other methods of wheat transformation previously described. Alternative Agrobacterium strains, such as AGL1 strain, or selective markers, such as genes encoding hygromycin resistance, can also be used in this method.

Example 4. Analysis of wheat transformants

Transformation was determined by one or more of the following methods, such as PCR analysis for the presence of one or more transgenes. PCR analysis was performed on genomic DNA extracted from 1-2 cm ^{2 of} fresh leaves using the mini-preparation method described by Stacey & Issac (1994). PCR reactions were carried out, for example, using SBEIIa primers: 5'-CCCGCTGCTTTCGCTCATTTTG-3 '[SEQ ID NO: 9] and 5'-GACTACCGGAGCTCCCACCTTC-3' [SEQ ID NO: 10] designed to amplify the fragment (462 p. n.) from the SBEIIa gene; or primers SBEIIb-DupFor 5'-AGATGTGAATGGCTGCTTGCTG-3 '[SEQ ID NO: 11] and SBEIIb-DupRev: 5'-CAGGTCGACCATATGGGAGAGC-3' [SEQ ID NO: 12] for SBEIIb (505 bp) The following conditions were used for the reaction: “hot start” (94 ° С, 3 min), then 30 denaturation cycles (95 ° С, 30 sec), annealing (55 ° С, 30 sec), elongation (73 ° С, 2 min), and then 1 cycle at 73 ° C (5 min).

Southern blot hybridization analysis was performed on DNA by large-scale (9 ml) extraction from lyophilized ground tissue (Stacey & Issac, 1994). The DNA samples were adjusted to a concentration of 0.2 mg / ml and hydrolyzed by restriction enzymes such as Hind III, Eco RI and Kpn I. Restriction enzyme hydrolysis, gel electrophoresis, and vacuum blotting were performed as described by Stacey & Issac (1994). Digoxygenin-labeled probes, including the intron region of 3 dc-SBEII constructs, were produced by the PCR method of McCreery & Helenjaris (1994). Hybridization of probes with Southern blots and detection by chemiluminescence was carried out by McCreery & Helenjaris (1994).

The results of PCR analyzes were systematized in Table 2. Plants that were transgene-positive, as demonstrated by PCR, included 27 independent transformants for DC-SBEIIa and 61 independent transformants for DC-SBEIIb.

table 2
Wheat transformation using constructs containing duplex SBEIIa and SBEIIb RNA Experiment No. No. of inoculated embryo No. of regenerated lines PCR positive lines DC-SBEIIa design 44 242 one one fifty 169 3 3 52 158 3 3 58 163 2 2 61 195 one one 72 185 one 0 83 241 one one 84 242 one one 85 153 5 5 109 262 13 10 Total 2010 31 27 DC-SBEIIb design 48 291 one one 51 166 one 0 53 194 one 0 55 261 one one 59 253 one 0 60 175 four 2 62 199 one 0 70 152 one 0 73 238 2 2 75 151 2 2 76 150 one 0 77 150 2 2 81 134 one one 87 230 5 3 92 233 8 5 110 240 29th 16 Total 3217 61 35

Example 5. Analysis of grain obtained from plants transformed with constructs containing duplex RNA

Morphology of starch granules

The morphology of starch granules originating from mature T1 seeds obtained from transformed T0 wheat plants was examined under an optical microscope. Ten individual grains from each of 25 T0 plants that were independently transformed with ds-SBEIIa and 12 plants that were independently transformed with ds-SBEIIb were analyzed. Each endosperm was carefully crushed to release starch granules that were dispersed in water and visualized under an optical microscope. Of the 25 ds-SBEIIa lines analyzed, 12 had grain with deformed starch granules (see, for example, FIG. 4), although visual observation revealed different levels of deformation in different seeds. In contrast, none of the 12 ds-SBEIIb lines showed a noticeable deformation of starch granules in the endosperm when observed under an optical microscope. The results are systematized in tables 3 and 4.

Table 3
Morphology of starch granules of T1 seeds obtained from ds-SBEIIa transgenic wheat lines Slide No. Line No. Morphology of starch granules * one 44.1a + 2 50.1b - 3 50.2b + four 50.3x - 5 52.1a + 6 52.2a + 7 52.3a +/- 8 58.1a - 9 58.2a - 10 61.2a - eleven 83.1b + 12 84.1a +/- 13 85.1a +/- fourteen 85.2c - fifteen 85.3a - 16 85.4b + 17 85.5a - eighteen 109.1a - 19 109.2c + twenty 109.3b + 21 109.4e - 22 109.7b - 23 109.8c - 24 109.10a + 25 109.11x + * The morphology of starch granules of 10 seeds of each line was observed. + means that all ten seeds have a normal morphology of starch granules; - means that there are seeds that have been severely deformed; and +/- means that there is some anomaly, that is, at least some seeds have a slight, but not strong deformation.

Table 4
Morphology of starch granules of T1 seeds obtained from ds-SBEIIb-transgenic wheat lines Slide No. Line No. Morphology of starch granules one 48.1a + 2 55.1a + 3 60.1a + four 60.4a + 5 73.1f + 6 75.1c + 7 75.3x + 8 77.1c + 9 77.2c + 26 110.16b + 27 110.17b + 28 110.18a + + means that all ten seeds from each line have a normal morphology of starch granules.

Observation of starch granules in polarized light revealed a significant decrease in birefringence in deformed starch granules (Fig. 5) for ds-SBEIIa-transformed grain. The absence of birefringence was observed in 94% of the starch granules in the seeds of line 50.1b, which correlated with their deformed phenotype, while normal starch granules of other seeds of the same line showed complete birefringence (table 5). It is assumed that seeds with normal starch granules are segregants that lack a transgene, and therefore they have a normal phenotype.

Table 5
Birefringence (DL) of starch granules of T1 seeds obtained from dc-SBEIIa transgenic wheat lines 50.1b Seeds from line 50.1b Microscopic field The number of starch granules that do not have DL The number of starch granules having a partial DL The number of starch granules having a full DL Seeds with deformed starch granules one 55 2 one 2 73 one 0 3 44 one 0 four 92 3 2 5 46 2 7 6 46 2 3 Total 356 (93.7%) 11 (2.9%) 13 (3.4%) Seeds with normal starch granules one one 3 110 2 one one 38 3 3 four 90 four 0 3 61 5 3 3 59 6 one 0 thirty Total 9 (2.2%) 14 (3.4%) 388 (94.4%)

The results of optical microscopy were confirmed by scanning electron microscopy (SEM) of starch granules. To do this, gold was sprayed onto the purified starch and scanned at 15 kV at room temperature.

Mass of grain

Individual grains obtained from ds-SBEIIa-transformed plants grown under equivalent conditions in a greenhouse were weighed (Table 6). Grains having severely deformed starch granules and obtained from plants 50.1b, 58.2a, 61.2a and 109 did not show a significant decrease in average weight compared to the grain of wild-type plants grown under the same conditions. It follows that the level of starch production is not significantly reduced even in seeds with severely deformed starch granules. These data also suggest that the yield of wheat grown in the field and having reduced SBEIIa activity in the endosperm should be almost within normal limits.

Table 6
Grain mass from T1 seeds obtained from ds-SBEIIa-transgenic wheat lines Transgenic line Seed number Seed mass (mg) Morphology of starch granules Trance
gene line No.
seed Seed mass (mg) Morphology of starch granules * 50.1b one 16.9 + 61.2a one 50.7 + 2 49.8 + 2 49.0 +/- 3 46.9 - 3 49.8 - four 50,0 - four 47.0 - 5 45.4 - 5 48.6 - 6 42.6 - 6 46.2 - 7 39.9 +/- 7 42,2 + 8 41.0 + 8 50,4 - 9 39.5 - 9 39.7 - 10 37.0 +/- 10 46.3 - 58.2a one 44.0 - 109.7b one 40.1 - 2 37,4 + 2 34.6 - 3 48.8 - 3 43.7 - four 43,2 + four 38.8 - 5 46.2 - 5 33.8 +/- 6 42.1 + 6 31.1 +/- 7 43.5 +/- 7 35.9 + 8 45.7 - 8 44.3 +/- 9 38.8 - 9 37.7 - 10 38.1 +/- 10 41,4 - + normal starch granules; - severely deformed starch granules; +/- slightly deformed starch granules.

Analysis of proteins SBEIIa and SBEIIb in the endosperm of transgenic wheat T2

Seeds (T2) from 13 dc-SBEIIa-transformed T1 plants, representing 5 independently transformed lines, and from 9 dc-SBEIIa-transformed T1 plants, representing 3 independently transformed lines, were analyzed for expression of SBEIIa and SBEIIb proteins in endosperm using electrophoresis in non-denaturing PAGE and Western blotting. All dc-SBEIIa plants were descended from lines having an abnormal morphology of starch granules, and all dc-SBEIIb plants were descended from lines having a normal morphology of starch granules, as described above. The antibody used to detect SBEIIa was a 3KLH rabbit antibody produced against a synthetic peptide having the amino acid sequence AASPGKVLVPDESDDLGC [SEQ ID NO: 13] corresponding to the N-terminus of mature SBEIIa, and 1: 5000 was diluted before use. The antibody used to detect SBEIIb was an R6 antibody produced against a synthetic peptide having the amino acid sequence AGGPSGEVMIGC [SEQ ID NO: 14] corresponding to the deduced N-terminus of mature SBEIIb, and this antibody was diluted 1: 6000 before use. The “second” antibody used was horseradish peroxidase conjugated anti-GAR antibody (1: 3000 dilution). Immunoreactive bands were detected using the Amersham ECL detection system.

Endosperms from each of the seven developing grains (15 days after flowering) of each of the 22 T1 plants were analyzed, since it was assumed that some of these plants could be heterozygous for the transgene. Twelve of the 13 ds-SBEIIa-transformed plants produced T2 progeny, indicating a decrease in the levels of SBEIIa protein in the endosperm. Obviously, all seven seeds from one line (50.3 × 0.9) completely lacked SBEIIa, and all seven seeds from four other plants showed a clear decrease in SBEIIa expression levels (Table 7). These plants are transgene homozygous lines. Seven lines showed segregation by the absence of SBEIIa or by lower levels of SBEIIa, but in some cases no decrease in protein level was observed, and these lines are probably homozygous for the transgene. The thirteenth line (50.3 × 0.6) was homozygous for wild-type expression (Table 7).

Table 7
Western blot analysis of endosperm proteins from ds-SBEIIa transgenic wheat lines T2 Transgenic line Target gene Morphology of T1 starch granules Segregation of the SBEII Protein Strip in T2 Grain 50.3x.6 SBEIIa + wild type homogeneous (+) 58.1a.3 '' - segregated by +/- and - 58.1a.7 '' - segregated by +, +/- and - 58.1a.9 '' - segregated by +, +/- and - 50.1b.3 '' - uniform in +/- 50.1b.4 '' - segregated by +/- and - 50.1b.5 '' - uniform in +/- 50.1b.9 '' - segregated by + and +/- 50.3x.9 '' - uniform in - 61.2a.8 '' - uniform in +/- 61.2a.9 '' - segregated by +/- and - 61.2a.10 '' - uniform in +/- 85.2c.2 '' - segregated by +/- and - 110.16b.14 SBEIIb + wild type homogeneous (+) 110.16b.2 '' + uniform in - 110.16b.17 '' + uniform in + 110.16b.5 '' + uniform in - 110.16b.19 '' + uniform in - 110.17b.3 '' + segregated by +/- and - 110.17b.6 '' + segregated by + and +/- 110.18a.9 '' + segregated by +/- and - 110.18a.17 '' + segregated by +, +/- and -

Of the nine ds-SBEIIa transgenic lines tested, three lines (110.16b.2, 110.16b.5 and 110.16b.19) were homogeneous (i.e., not segregated) by the absence of SBEIIb expression in each of the seven offspring seeds, two lines were homogeneous in wild-type expression, and the remaining four lines showed segregation by lack of expression, by reduced expression, or by wild-type expression (Table 7). Seed embryos can be grown (“saving” the embryo) to produce T2 plants and T3 seeds, which are then screened by PCR and analyzed for protein expression to confirm the genetic status of T2 seeds with respect to the transgene.

These data showed that duplex RNA constructs provide an effective reduction in the expression level of SBEIIa and SBEIIb genes in wheat endosperm. These data also showed that a decrease in the expression level of the SBEIIb gene alone does not significantly affect the morphology of starch granules.

The expression of SBEIIb gene in transgenic seeds containing ds-SBEIIa transgene and not containing SBEIIa protein, and the expression of SBEIIa gene in seeds containing ds-SBEIIb were also analyzed using Western blot analysis. Surprisingly, transgenic seeds containing ds-SBEIIa were found to have much lower levels of SBEIIb. However, in seeds transgenic by ds-SBEIIb, the opposite effect was not observed. The expression of SBEIIa did not change in seeds in which SBEIIb was completely inactivated by ds-SBEIIb. It is possible that the expression of SBEIIb was suppressed by the construction of ds-SBEIIa, which is due to the homology of the gene sequences in the region used to construct the duplex, and it is also possible that the activity of SBEIIb was reduced by the action of the dc-SBEIIa transgene in accordance with some other mechanisms.

The expression levels of SBEIIa and SBEIIb genes can also be specifically determined at mRNA levels by standard methods, such as Northern hybridization or RT-PCR, for example, using probes from non-conserved regions or pairs of primers that hybridize with unique sites in one of these genes, but not in other areas, for example in 3'-untranslated areas. Such regions or sites can be easily identified by comparing the sequences of two genes.

Example 6. Analysis of starch obtained from transformed wheat

Amylose and Amylopectin Levels in Transgenic Wheat Grain

The amylose content of starch from the pooled pool of six T1 seed samples was determined as described in Example 1. Pooled seed samples were obtained from transgenic wheat lines, as described below.

Pool 1: seeds with deformed starch granules from dc-SBEIIa transgenic line 85.2c

Pool 2: seeds with normal starch granules from DC-SBEIIa-transgenic line 85.1a

Pool 3: seeds with normal starch granules from DC-SBEIIb-transgenic line 110.18a

Pool 4: pooling seeds with deformed starch granules from ds-SBEIIa transgenic lines 58.1a, 58.2a and 61.2a

Pool 5: seeds with normal starch granules from ds-SBEIIa transgenic line 83.1b

Pool 6: seeds with normal starch granules from dc-SBEIIb-transgenic line 75.3x

Each analysis of starch samples was performed in four replicates. In these analyzes, the following regression equation was used to convert the optical density to the amylose content: Y = 57.548x - 8.793, where Y is the amylose content (%) and x is the optical density.

The results are presented in table 8. The presence of deformed starch granules clearly correlates with an increase in amylose content. Starch from grain with deformed starch granules obtained from ds-SBEIIa transgenic lines (pools 1 and 4) had an amylose content of more than 50%, and other starch pools obtained from grain with normal starch granules had an amylose content in the range of 21 -26%. These pools included starch from line IIb 110.18a, which had a lower level of SBEIIb expression (Table 8), suggesting that inactivation of one SBEIIb gene in wheat does not significantly increase the levels of amylose in wheat starch.

Table 8
Amylose content in transgenic wheat lines determined by iodometric method Starch sample Transgenic line Amylose Content (%) Replication 1 Replication 2 Replication 3 Average Pool 1 85.2s 65.7 54,2 53,2 57.7 Pool 2 85.1a 23.7 22.5 26.7 24.3 Pool 3 110.18a 22.3 21.0 21.5 21.6 Pool 4 58.1
58.2a
61.2a 53.9 52.8 58.5 55.1 Pool 5 83.1b 26.5 25.3 24.8 25.6 Pool 6 75.3x 24.3 20.6 19.5 21.5

The second series of analyzes was performed by the iodometric method using a sample of pool 4 and wheat starch, which is deficient in SSII (Yamamori et al., 2000), and barley line M292, which is mutant in SSIIa . The amylose content determined for starch from a pool of 4 seeds of wheat (ds-SBEIIa transgenic lines) was significantly higher than the amylose content in starch obtained from SSII mutants of wheat and barley.

From this it follows that the content of amylopectin in the starch obtained from this grain, compared with the content of amylopectin in wild-type grain, significantly decreased from about 75% to less than 50% or even less than 20%.

Lines containing both dc-SBEIIa- and dc-SBEIIb transgene were generated by crossing the transgenic plants described above. The amylose content in the starch of the grain obtained from such offspring exceeded the amylose content in the starch of plants containing only dc-SBEIIa, for example, by 75 or 80%, which indicates that inhibition of SBEIIb, in addition to inhibiting SBEIIa, also leads to an increase in levels amyloses.

Example 7. Comparison of SBEIIa from genomes A, B and D

Construction of cDNA and wheat genomic libraries

cDNA and genomic libraries for wheat endosperm were obtained by standard methods in phage vectors (Sambrook et al., 1989). Two cDNA libraries were obtained, one from Rosella variety RNA (Rahman et al., 1999), and the other from Wyuna variety (Rahman et al., 2001). The library for the Rosella variety was created in the ZAPII vector using Eco RI and Not I primers, and the library for the Wyuna variety was created in the ZipLox vector (Life Technology) in accordance with the protocols attached to the supplied reagents. The titers of these libraries were 2 × 10 ⁶ b.p. and were tested using E. coli strain Y1090 (ZL). The genomic library was constructed from A. tauschii DNA cultivar 10097. The DNA was hydrolyzed with Sau 3A and ligated with the partially completed vector lambdaGEM12 (Promega). Cloned fragments can be isolated by hydrolysis with Sac I or Xho I enzymes . Genomic DNA libraries of T.aestivum have been described by Turner et al. (1999).

Isolation of SBEIIa cDNA Sequences

cDNA was isolated from a library obtained from Rosella cultivar using a probe for the wheat SBEI gene sequence under low stringency conditions (Rahman et al., 2001). The longest of the obtained clones, designated sbe9, was sequenced and it was found that it encodes a sequence of type SBEIIa (GenBank AF338432.1). Then, three clones were isolated from the cDNA library of the wheat endosperm of the Wyuna variety (Rahman et al., 2001) using a probe corresponding to the positions 536-890 of the sbe9 clone. The library was screened under the following hybridization conditions: 25% formamide, 5 × SSC, 0.1% SDS, 10 × Denhardt's solution, 100 μg / ml salmon sperm DNA, at 42 ° C for 16 hours, followed by washing in 2 × SSC , 0.1% SDS, at 65 ° C for 1 hour (3 ×) (medium hardness). Sequencing the clones, three different sequences were obtained, which are presented below as sr995 and sr997 (Fig.6).

The study of these cDNA sequences showed that different sequences were expressed in the wheat endosperm, and these sequences probably correspond to sequences of SBEIIa transcripts from different wheat genomes. Comparison of these sequences with other known wheat SBEIIa cDNA sequences using the PILEUP program showed that the sr995 and sr996 sequences form clusters with the mRNA sequence derived from the wSBE-D1 D genomic sequence (sr854) (Fig. 7), which suggests that sr995 and sr996 are transcripts originating from SBEIIa of genome D. The sequence sr997 formed a cluster with the sequence Y11282 (Nair et al., 1997), which indicated that they probably originated from the same genome, or A or B. The previously described clone sbe9 (AF338432.1) probably comes from the same genome as Y11282, but is an alternative splicing variant, as a result of which one exon was excised near the 5'-end.

Identification of SBEIIa genes from wheat genomes A, B and D T.aestivum

Differences in the sequences of genes or RNA transcripts can be taken as a basis for the design of primers specific for genome A, B, and D for screening for mutations either at the gene level or at the RNA level. So, for example, Fig.6 compares the nucleotide sequences of SBEIIa from cDNA, including the GenBank sequence reg.No. Y11282, and incomplete cDNA sequences of clones sbe9 (AF338432.1), sr997 and sr995. Genomic sequences for T. aestivum SBEIIa genes are shown, for example, in Table 1. These genomic sequences were assigned to genomes A, B, and D. Comparison indicated polymorphism, each of which could be used to identify sequences by molecular methods.

To identify products from genomes A, B, and D, a direct primer designed based on the exon 5 region (5'-ATCACTTACCGAGAATGGG-3 ') [SEQ ID NO: 15] and a reverse primer designed based on the sequence in exon 6 ( 5'-CTGCATTTGGATTCCAATTG-3 ') [SEQ ID NO: 16]. Such primers can be used in PCR reactions for screening varieties or lines that are mutant for one or more SBEIIa genes originating from genomes A, B or D (see below).

PCR markers were also obtained to identify SBEIIb genes from wheat genomes A, B or D. For example, PCR reactions using a pair of primers ARA19F (5'-CACCCATTGTAATTGGGTACACTG-3 ') [SEQ ID NO: 17] and ARA15R (5'-TCCATGCCTCCTTCGTGTTCATCA-3') [SEQ ID NO: 18] and subsequent hydrolysis of the amplification restriction enzyme Rsa I products allow the identification of SBEIIb genes from three genomes.

Differences in cDNA sequences are reflected in the deduced protein sequences. So, for example, the deduced full-length amino acid sequences for the polypeptides of genome D (sr854) and genomes A or B (Y11282) were compared, as shown in Fig. 8. Significant differences were observed in regions 688-698 and 735-6, which can be used to produce genome-specific antibodies against SBEIIa proteins for screening wheat varieties that do not have one or more genome-specific activities. Other differences were observed in transport peptide sequences that correspond to amino acid positions 1-54 in FIG.

Example 8. Identification of wheat varieties having mutations in one or more SBEII genes .

Identification of silent mutations of SBEIIb in genomes B and D

A total of 1,500 wheat lines, including 300 Australian wheat varieties, 900 wheat lines taken from the Australian Winter Crop Collection (AWCC, Tamworth, NSW Australia), and 300 local wheat varieties were screened by PCR amplification of an SBEIIb marker corresponding to the intron polymorphic region 3 using primers ARA19F (see above) and ARA23R (5'-CTGCGCATAAATCCAAACTTCTCG-3 ') [SEQ ID NO: 19]. PCR amplification conditions are described above. The amplification products were hydrolyzed with the restriction enzyme Rsa I and subjected to polyacrylamide gel electrophoresis. In three lines (Aus12745, Aus17340 and Aus10103) there was no marker of genome D, and in two lines (Aus12509 and Aus12565) there was no marker of genome B (Fig. 9). These lines represent putative null mutants in SBEIIb genes from genomes B or D.

To confirm the results of PCR, analysis was performed by Southern blot hybridization on DNA from null mutant lines. Hind III-hydrolyzed DNA obtained from plants by standard methods was subjected to electrophoresis on 1% agarose gels and blotted on a Hybond N + nylon membrane (Amersham). Radiolabeled probes were generated from the intron 3 region of the SBEIIb gene (positions 2019-2391, see FIG. 2) using the Megaprime DNA tagging system (Amersham Pharmacia Biotech UK Ltd) and used for hybridization under stringent conditions. Aus17340 and Aus10103 did not contain a band of ~ 4.8 kb. genome D, while Aus12509 and Aus12565 did not contain a band of ~ 3.4 kb. genome B (figure 10). It was confirmed that these lines are null mutants for SBEIIb genes of genome D or B, respectively.

Generation of double null mutants B + D

The following crosses were received:

Aus17340a x Aus12509

Aus17340b x Aus12509

Aus17340a x Aus12565

Aus17340b x Aus12565

Aus12745 x Aus12509

Aus12745 x Aus12565

Aus17340a and Aus17340b are two different biological types of the same cultivar Aus17340, and it was confirmed that both of them are null mutant for the SBEIIb marker gene in genome D. F1 plants were self-pollinated and F2 progeny were screened by PCR for plants that were mutant for SBEIIb genes in genome B and D (double null mutants). Segregation by SBEIIb mutations was observed by PCR amplification using pairs of primers AR2b19cF (5'-CTATGCCAATTGAACAACAATGC-3 ') [SEQ ID NO: 20] and AR2b23cR (5'-CGTGTTCATCAATGTCTAC] (which amplify the same region as ARA19F / ARA23R) followed by hydrolysis with the restriction enzyme Rsa I. A typical segregation pattern is shown in FIG. 11. The analysis using the chi-square criterion revealed that the pattern of segregation of crosses Aus17340a x Aus12509 and Aus17340a x Aus12565 corresponds to the expected ratio of 9: 3: 3: 1 (table 9). Segregation in other crosses has been greatly altered.

Table 9
Analysis of the F2 population for crosses between null mutants of SBEIIb in subgenomes B and D, performed using the chi-square test Cross / Phenotypes 17340a
x
12509 17340b
x
12509 17340a
x
12565 17340
x
12565 12745
x
12509 12745
x
12565 normal 85 63 56 72 95 21 null mutation in B 38 39 25 35 eleven 2 null mutation in D 23 29th eleven eleven 57 16 null mutation in BD 6 10 four 6 3 0 Total 152 141 96 124 166 39 χ ² _{(9: 3: 3: 1)} 5.52 9.73 6.19 12.91 39.79 16.66 Tabulated value χ ² _{(9: 3: 3: 1)} (0.05), df3 = 7.81

Albino plants were detected in all crosses, regardless of parental lines, as a result of which it was found that the mutant gene related to the chlorophyll gene was also segregated in these populations. Of the 24 albino plants analyzed, 23 plants had B + D double null mutations, and one plant was obviously of the wild type. Five normal green-looking plants with B + D double null mutations were identified from 718 tested lines. Three of them came from the cross Aus17340b x Aus12509 (BD219, BD303, BD341), one plant came from the cross Aus17340a x Aus12509 (BD54), and one from Aus17340b x Aus12565 (BD636). The results showed that mutations in SBEIIb genes of genome B and D were closely linked to a mutation in a gene related to the chlorophyll gene, which gave the albino phenotype in case two mutated loci were transmitted. However, recombination events between the SBEIIb gene and a gene related to the chlorophyll gene were identified, which led to the production of normal lines with B + D double null mutation, albeit with a very low frequency. This indicates that these two genes, although closely linked, can be separated.

Example 9. SBEIIa and SBEIIb are linked in a wheat plant

Isolation of BAC clones

A large insertion cosmid library (BAC) constructed from A. tauschii meyeri wheat (Moullet et al., 1999) was probed with the intron 3 region of the SBEIIb gene (positions 2019-2391, FIG. 2) to isolate BAC containing the SBEIIb gene . Four positive clones were identified that designated BAC-4, -5 and -9 and -12. To confirm that they contain the SBEIIb gene, the DNA of these clones was extracted, hydrolyzed with Hind III or Eco R1 enzymes, and Southern blot hybridization was performed using the same probe (Fig. 12). Clone BAC-5 detected one strong hybridizing band ~ 7.5 kb in size. with EcoR 1 and four bands of sizes ~ 6.1, 3.6, 2.3 and 1.7 with Hind III (FIG. 12). This indicated the presence of SBEIIb at BAC-5. For analysis of the presence of the 3'-region of the gene on BAC-5 on this clone, PCR amplification was performed using specific primers designed on the basis of SBEIIb cDNA sequence for amplification of exons 17 (AR2b3pr2F, 5'-GGATATGTATGATTTCATGG-3 ') [SEQ ID NO: 22] and AR2b3pr2R, 5'-CCATAAAGTTAAGATAACCC-3 ') [SEQ ID NO: 23]) and 20 (AR2b3pr1F, 5'-GACATCAGACCACCAGTACG-3') [SEQ ID NO: 24] and AR2b3pr1R, 5'-CTTAG -3 ') [SEQ ID NO: 25]). Both primer pairs amplified the expected 128 bp products. for exon 17 and a size of 145 bp for exon 20, which indicated that BAC-5 contained the 3'-end of the SBEIIb gene. This was further confirmed by sequencing the PCR product from exon 20.

BAC-5 was also tested for the presence of the SBEIIa gene in addition to the SBEIIb gene. The nucleotide sequencing reactions using the primer AR2akpnIF 5'-GGTACCGCAGAAAATATACGAGATTGACCC-3 '[SEQ ID NO: 26] revealed a sequence corresponding to the region of intron 3 of the SBEIIa gene, and this sequence was similar to the sequence extending from position 2265 to position 2478 (figure 1) gene w SBEII-D1 . This result suggests that SBEIIa is also present on BAC-5, which means that the SBEIIa and SBEIIb genes are probably closely linked in a wheat plant.

In Situ Hybridization Fluorescence (FISH)

In situ hybridization with the genomic F2 clone wSBEII-D1 (Rahman et al., 2001) and with the clone wSBEII-D2 (Rahman et al., 2001) was performed using the squeezed preparation method on Aegilops tauschii and wheat chromosomes described by Turner et al. (1999). The identity of the hybridized chromosome was verified by double-labeling pSc119.2, a repeating sequence used to identify chromosomes (Mukai et al., 1990). Both wSBEII clones hybridized with the proximal region of chromosome 2 (Fig.13), indicating the proximity of two SBEII genes in wheat.

Wheat SBEIIb null mutants are also SBEIIa mutants

SBEIIb null mutants identified as described above were screened for mutations in the SBEIIa gene using Sr913F primers (5'-ATCACTTACCGAGAATGGG-3 ') [SEQ ID NO: 27] and E6R (5'-CTGCATTTGGATTCCAATT-3 SEQ ID NO: 28]. These primers were designed to amplify the intron 5 region of the wSBE II-D1 gene and to identify SBEIIa genes on genomes A, B, and D.

It was found that null mutants of the SBEIIb gene , Aus12565 and Aus12509 in genome B are also null mutants of the SBEIIa gene in genome B. Similarly, null mutants of SBEIIb in genome D, Aus17340 and Aus10103 are also null mutants of SBEII genome D. In addition, the double mutant SBEIIb lines in the B + D, BD341, and BD636 genomes also represent double null mutants of the SBEIIa gene in the B + D genome. The data obtained confirm that in a wheat plant, unlike rice and corn, the SBEIIa and SBEIIb genes are closely linked to each other and this indicates that mutations of copies of these genes in the B and D genomes described above are deletion mutations.

Wheat plant with three null mutations of the SBEIIa gene

The methods described above can be used to isolate SBEIIa and / or SBEIIb mutants of genome A. Thus, for example, BAC-5 regions closely linked to SBEIIa and / or SBEIIb were used as probes or to design PCR primers for screening for mutations in genes of genome A. Mutants in genome A were crossed with lines having double null mutations in B + D genomes to produce a line with triple null mutations in A + B + D genomes. Alternatively, mutation of the double null mutants of the B + D genome was carried out by irradiation or other methods, after which a triple null mutant was identified in which SBEIIa activity was completely absent, and sometimes SBEIIb activity was absent. Thus, a non-transgenic wheat variety with a very high level of amylose was obtained.

Example 10. Mutation of the SBEIIA gene in wheat

Mutation of the SBEIIa gene in wheat, leading to a decrease in the activity of the SBEIIa gene, can be achieved either by irradiation with gamma rays or by chemical mutagenesis, for example, using ethyl methanesulfonate (EMC). To create a mutation induced by gamma radiation, the seeds were irradiated at a dose of 20-50 kP supplied from a ⁶⁰ C source (Zikiryaeva & Kasimov, 1972). EMC mutagenesis was performed by treating the seeds with ethyl methanesulfonate (EMC) (0.03%, v / v) as described by Mullins et al., (1999). Mutant grains with a double null phenotype in the B + D genome were identified by an increased amylose content or by an altered morphology of starch granules and confirmed by the methods described above. Mutants in SBEIIa that retain SBEIIb activity can be re-mutagenized and their progeny can be screened for the absence of SBEIIb activity in addition to the lack of SBEIIa activity, or the SBEIIa mutant can be crossed with the SBEIIb mutant to combine mutations and to produce non-transgenic wheat variety, in the endosperm of which, mainly, SBEII activity is absent.

Example 11. Wheat mutants SGP-1 have reduced SBEIIa- and SBEIIb activity

Genes for starch synthase II (SSII) in wheat genomes A, B and D ( Triticum aestivum ) encode 100-105 kDa polypeptides, which are also known as starch granule proteins-1 (SGP-1). SSII (SGP-1) consists of three polypeptides with a molecular weight of approximately 100, 104 and 105 kDa, which are encoded by a homologous series of genes located on the short arm of chromosomes 7B, 7A and 7D, respectively (Denyer et al., 1995, Yamamori & Endo, 1996). Yamamori et al. (2000) obtained SGP-1-free wheat by crossing lines that lacked SGP-1 protein forms specific for genome A, B, and D, as analyzed by protein electrophoresis. Evaluation of seeds that do not contain SGP-1 showed that such a mutation leads to a change in the structure of amylopectin, to an increase in the amylose content and to the deformation of starch granules (Yamamori et al., 2000). In addition, experiments performed on mature grain using electrophoresis revealed that levels of SBEII (SGP-2) and SSII (SGP-3) associated with starch granules were significantly reduced. The molecular mechanisms of mutation (s) leading to their production of lines lacking SGP-1 are unknown.

The authors conducted experiments to further characterize the wheat line, in which SGP-1 is completely absent in starch granules obtained from mature grain. In order to determine if SSII genes are present in each of the wheat genomes A, B, and D that do not contain SGP-1, the DNA was extracted from wheat that did not contain SGP-1 and from wild-type wheat (control) of the Chinese spring, and then it was analyzed by PCR using a combination of ssIIa primers (5'-CCAAGTACCAGTGGTGAACGC-3 ') [SEQ ID NO: 29] and ssIIb (5'-CGGTGGGATCCAACGGCCC-3') [SEQ ID NO: 30] for genome B or ssIIa and ssIIc (5'-CATGTGAGCTAGCTTTCGCCC-3 ') [SEQ ID NO: 31] for genomes A and D. The amplified region was between positions 2472 and 2821 bp wSSIIA (GenBank, Reg. No. AF155217) or the corresponding regions of wSSIIB or wSSIID. The amplified region consisted of a part of exon 8 and was chosen because it allows us to clearly identify the products of genomes A, B, and D. Amplification was carried out under the following conditions: 35 cycles at 94 ° C, 30 sec; at 60 ° C, 1 min and at 72 ° C, 2 min, the PCR fragments produced from genomes A, B and D of wheat without SGP-1 had the same size as the corresponding fragments obtained from wheat of the Chinese variety spring. PCR amplification of the segments of isoamylase genes and SSI genes, which are starch biosynthesis genes closest to the SSII starch biosynthesis genes and located on both sides of SSII (Li et al., 2002), showed that these genes can be amplified from each genome A, B and D of wheat that does not contain SGP-1. Therefore, the null phenotype of SGP-1 was not caused by a deletion of any of these genes on the short arm of chromosome 7.

Evaluation by scanning electron microscopy showed that starch granules obtained from developing wheat seeds that did not contain SGP-1 in the growth phase, starting 10 days after flowering and lasting until they mature, were markedly deformed. The distribution of chain lengths of unbranched starch in the mutant, evaluated by capillary electrophoresis, indicated an increase in the number of shorter chains (to DP 8) and a decrease in the number of longer chains of DP 9-22.

Expression of starch synthases and branching enzymes in SGP-1 endosperm

The expression of starch synthases and branching enzymes in starch granules was studied in plants not containing SGP-1, and compared with the expression level in wild-type wheat of the Chinese Spring variety. Regardless of the stage of seed development, there was a significant decrease, by about 90-96%, of the amount of SBEII and SSI in the starch granules of the line containing no SGP-1, as well as the absence of SSII (Fig. 14). The use of specific antibodies showed that the SBEII strip obtained from starch granules of Chinese spring wheat varieties consisted of approximately 1: 3 SBEIIa and SBEIIb. In the null mutant of SGP-1, this amount was so low that its relative content could not be determined using antibodies. There was also a decrease in the level of HBSS I at an early stage of grain development. Obviously, the SGP-1 mutant had a reduced level of polypeptides associated with starch granules, including SBEIIa and SBEIIb. A decrease in the number of polypeptides associated with starch granules (SBEIIa and SSI) was not observed in the grain of wheat lines used to produce the null mutant according to SGP-1 (Yamamori et al., 2000), and it was suggested that this effect was in particular, due to the lack of SSII.

Branching enzymes and starch synthases were also analyzed in the soluble phase of the developing endosperm. The relative amounts of soluble SBEIIb were similar in the Chinese spring wheat line and in the wheat line not containing SGP-1, however, a decrease in the amount of SBEIIa was observed in the soluble phase of the mutant (Fig. 15). However, this may be partially due to the genealogy of the line that does not contain SGP-1.

These data demonstrated that SBEIIa activity may decrease pleiotropically due to a mutation in the SSII gene. Although a mutation in only one SSII gene gave relative amylose levels in starch of less than 50%, it is believed that mutations in other genes, that is, not SBEIIa , can be combined with SBEII mutations with increasing levels of amylose and producing modified starch.

Example 12. Multiple SBEI Deficient Isoforms

Purification of starch-branching wheat enzymes using anion exchange chromatography revealed three activity peaks (FIG. 16, Morell et al., 1997). Extracts of endosperm from Chinese spring wheat (CS) showed the presence of four SBEI polypeptides on non-denaturing PAGE, as was shown using a polyclonal anti-WBE-1 antibody produced against a synthetic peptide with the amino acid sequence corresponding to the N-terminal sequence of peak 1 protein (FIG. .16B). Analysis of CS nullisomic-tetrasomic lines showed that these polypeptides are encoded on chromosome 7, and the bands on the immunoblot were assigned to genomes A (lane A), B (lane B) and D (lanes Di and Dii), and their activities were named A- major, B major and D major activities, respectively. Immunoblot analysis of purified fractions representing activity peaks obtained by anion exchange chromatography showed that the first peak contained A major and D major SBEI activities, and the second peak contained B major SBEI activity (Fig. 16C).

The localization of the gene encoding the major SBEI activity on chromosome 7 corresponded to a certain localization of the three well characterized and related genes w SBEI-D2 , w SBEI-D3 and w SBEI-D4 . The deduced sequence of the SBEI major protein showed that it is encoded by the last of these genes w SBEI-D4 (Rahman et al., 1997, Suzuki et al., 2003). The presence of the fourth SBEI gene was predicted from Southern blot hybridization data (Suzuki et al., 2003).

Identification of null mutations of the major protein gene SBEI

To identify null mutations in which there was no expression of one or more SBEI isoforms, the selected germplasm pools of wheat were screened by immunoblot detection of the SBEI major protein after electrophoresis in a non-denaturing gel. In this case, the anti-wSBEI antibody described above was used. Of 182 Australian wheat hexaploid lines analyzed, 13 lines were identified as lines not expressing major SBEI-D, 16 lines did not contain major SBEI-B, 10 lines did not contain SBEI-A, and two lines (Bindawarra and Vectis) did not contain both isoforms A and B. These lines were considered as lines containing null mutations of the corresponding SBEI genes in the genome. The frequency of null mutations in the gene for major, SBEI (~ 23%) was similar to the frequency of null mutations in the GBSS gene (22%) (Boggini et al., 2001).

Generation of a line with triple nullation according to the major SBEI gene

Using immunoblot analysis, it was found that the varieties Bindawarra and Vectis did not have A-major and B-major SBEI activities, and the variety Cadoux was identified as a variety without D-major activity. A 185-line F2 progeny population obtained by crossing Vectis x Cadoux was screened by immunoblotting. However, no lines were obtained in which all three activities would be absent, which suggests that each of these offspring had low viability or showed a certain interaction between the genomes. Therefore, the hereditary line VC3.1.11, in which B- and D-major activities were absent, was crossed with the Chinese Spring strain line, which had a chromosome modified by genetic engineering (CS7AL-15) and in which A-major activity was absent. Double haploid lines were screened by PCR using primers ARBE1CF (5'-GGGCAAACGGAATCTGATCC-3 ') [SEQ ID NO: 32] and ARA9R (5'-CCAGATCGTATATCGGAAGGTCG-3') [SEQ ID NO: 33] and 2 lines (A113 and D13) from 160 lines showed a complete absence of SBEI-major activity, as determined by immunoblotting in non-denaturing gels. 17 shows a representative pattern of segregation of double haploid lines, including A113 (lane 6).

Endosperm line A113 was evaluated for residual SBE activity. The wild-type wheat variety, D28, showed two peaks of SBEI activity. In contrast, A113 extracts gave the first peak of activity, however, the second peak of activity was completely absent. Amino acid sequences obtained from the purified fraction containing such activity indicated the presence of an SBEI-type protein in A113. However, this fraction did not detect a reaction with the anti-WBEI antibody in a non-denaturing gel. The branching activity in A113 corresponded to a ~ 80 kDa protein, which may be an SBEII type enzyme because it cross-reacted with antibodies against potato SBE and against maize SBEII.

These data demonstrate that mutant SBEI lines can be generated in wheat. The combination of SBEI mutations with SBEIIa mutations and, optionally, SBEIIb mutations led to the production of wheat plants whose grain contained starch with very high levels of amylose.

Example 13. Identification of mutant wheat lines containing chromosome 2A with a mutation in the SBEII gene

In an attempt to identify a wheat line having a mutation in SBEIIa or SBEIIb genes , 2,400 wheat hexaploid lines were screened for null mutations by SBEIIb in genomes A, B or D. In PCR reactions on genomic DNA samples of wheat plants from each line, primers AR2b19cF / AR2b23cR, after which the amplified products were hydrolyzed by the Rsa I enzyme and gel electrophoresis. This marker amplified the intron 3 region (nucleotide positions 2085-2336 in the wheat SBEIIb gene, FIG. 2) and was specific for SBEIIb . This screening allowed the identification of three null mutants by SBEII in genome D and two null mutants by SBEII in genome B, as described above in the examples. No mutant lines were found in which the genome A band corresponding to SBEIIb was absent . This suggests that wheat lines containing chromosome 2A with the mutant SBEIIb gene are not found in nature.

A mutant population of wheat induced by gamma radiation ( ⁶⁰ Co-source) and generated by Tony Prior & Rohit Mago (CSIRO) was used to screen for induced mutations in the SBEII gene of wheat. This wheat population was generated from the offspring of F2 cross-country Gabo 1BL.1RS x Veery 3. In total, 2694 mutant seeds of this population were screened, as described above in PCR reactions using AR2b19cF and AR2b23cR primers. Two seeds, designated MLT2B8 and MLT2D1 and originating from the same plant, were identified as not containing the SBEIIb allele in genome A (Fig. 18). Seeds containing null mutations of SBEIIb in genomes B or D were not identified in this population.

As shown in the examples described above, the SBEIIa and SBEIIb genes were closely linked in a wheat plant on the long arm of chromosome 2. Accordingly, the authors tested the DNA of these seeds for the presence or absence of the SBEIIa gene in genome A by PCR reactions using Sr913F primers / E6R. These primers amplify the intron 5 region of wSBEII-D1 (nucleotide positions 2959-3189, FIG. 1 [SEQ ID NO: 1]). After amplification, the products were subjected to electrophoresis in a 5% sequencing gel (ABI Prism DNA sequencer). Fluorescently labeled products were analyzed using a Genescan computer program. Scanning profiles showed that the amplified products of both mutant seeds MLT2B8 and MLT2D1 lacked a product corresponding to the SBEIIa gene in genome A, which indicated that both seeds had zero alleles in the A genome of the SBEIIa gene and also of the SBEIIb gene.

Null mutations in these seeds were further confirmed using genome A specific markers for SBEIIa , ARIIaAF (5'-GCAAAAGCCAGATCATAAATTTAGAGC-3 '[SEQ ID NO: 34] and ARIIaAR (5'-CTTCCAATTCATTGTTAATGGTCACAC-3') : 35], which amplify only the SBEIIa gene product in genome A (nucleotide positions 3024-3131 in wSBEII-DA1, Fig. 1). Although this pair of primers amplified 110 bp of the material from a Chinese spring plant, however this the product was clearly absent in these two seeds with the alleged mutation.This result was similar to the result obtained for negative to ntrolya dt2AS, which was a grade line of Chinese Spring, having a genetically engineered chromosome 2A in which the long arms was missing. Since genes SBEIIa and SBEIIb were localized on the long arm of chromosome 2, this line was absent allele of both these genes in the genome, and therefore this line can be used as a negative control (Fig.19).

Embryos from the mutant seeds MLT2B8 and MLT2D1 identified as mutants for SBEIIa and SBEIIb in genome A were cultivated to generate plants. The starch obtained from the seeds of these plants was analyzed for amylose content, chain length, and other properties in order to determine whether null mutations in both SBEIIa and SBEIIb genes in Genome A affect starch properties.

As described above, five lines were generated with a mutation in the SBEIIa and SBEIIb genes of genomes B and D. Of these, lines BD 219 and BD636 were cultivated in a greenhouse and crossed with null mutant lines A, MLT2B8 and MLT2D1. A double haploid population was generated from F1 seeds of these crosses and homozygous plants with three null mutations were obtained. Such triple null mutants should occur in double haploid populations with a frequency of 1 out of 8. Null mutations in genome A can be combined with either mutations in genome B or mutations in genome D by similar crosses. In other crosses, any of the null alleles can be introduced into any suitable genetic background to inform the plant of the desired agronomic or other characteristics.

To obtain durum wheat (such as, for example, the Wollaroi variety) with mutations in the SBEIIa and SBEIIb genes of genome A, the following crosses were performed:

1) Wollaroi x MLT2B8 or MLT2D1 for producing durum wheat with null mutation according to SBEIIa / SBEIIb in genome A with the Wollaroi phenotype,

2) A-genomic null mutant of wheat durum (Wollaroi) x B-genomic null mutant of SBEIIa / SBEIIb, to produce a double AB-null mutant of SBEIIa / SBEIIb wheat durum (Wollaroi),

Alternatively:

1) Wollaroi x B-genomic null mutant of the wheat line, for the production of the B-genomic null mutant of wheat durum (Wollaroi),

2) Wheat B-genomic null mutant Wollaroi x Wheat A-genomic null mutant to produce a double AB-null mutant for durum SBEIIa / SBEIIb wheat.

Such crosses make it possible to generate durum wheat with a high content of amylose, the use of which also favorably affects health, as well as the use of hexaploid wheat with a high content of amylose.

Example 14. Confirmation of the high content of amylose in wheat grain by separation methods on a column with sepharose 2B

The amylose content in starch obtained from grain of transgenic wheat plants containing SBEIIa / SBEIIb- inhibiting gene constructs was determined by separation on a Sepharose column. In this method, starch molecules were separated on a column according to their molecular weight. The separated fractions were analyzed using a starch analysis kit (Sigma) in accordance with the instructions of the suppliers.

Approximately 10 mg of starch was dissolved in 3.0 ml of 1N NaOH (degassed) by incubation at ³⁷ ° C for 30 minutes. The starch solution was centrifuged for 15 minutes to precipitate insoluble components. The supernatant was loaded onto a Sepharose CL2B column at a pump speed of 1 ml / min. The column was run using 10 mM NaOH as buffer and fifty fractions of 2.5 ml each were collected. The pH of fractions 9-50 was adjusted to 4.5 by the addition of 35 μl of 1M HCl. An aliquot (250 μl) of each sample was transferred to a test tube, and then 250 μl of starch reagent (starch assay kit, Sigma) was added. The control included a starch analysis reagent containing only starch reagent (250 μl) and water (250 μl); a glucose analysis control reagent containing only 500 μl of water; a control sample containing only 250 μl of a starch sample and 250 μl of water; and a test sample containing only 250 μl of starch reagent and 250 μl of starch sample. The samples and controls were incubated at 60 ° C for 60 minutes, then 200 ul of each was transferred to a new tube followed by addition of 1 ml of glucose reagent (kit for analyzing starch, Sigma) and incubated at ³⁷ ° C for 30 minutes. The optical density at 340 nm was used to determine the amount of starch (mg) in each fraction in accordance with the instructions attached to the kit.

The chromatogram of starch samples revealed two peaks eluted from a Sepharose column. The amylose content (second peak) of each sample was calculated as a percentage of the total starch content at both peaks.

Using this method, it was determined that the amylose content in the dc-SBEIIa transgenic Acc line. 144087, which is homozygous for the transgene, is 78%, and the amylose content in the dc-SBEIIb transgenic line Acc.144008 (homozygous transgenic line of transformant IIb 110.16b) is 23% (Fig. 20). Compared with this method, the iodometric method gives an amylose content for these lines of 88.47% and 27.29%, respectively (table 10).

Functional properties, such as gelatinization temperature, paste viscosity and starch swelling volume, were analyzed using differential scanning calorimetry (DSC) on a high-speed viscosity analyzer (RVA) and using a starch swelling test, respectively. The structure of such starch was analyzed using x-ray crystallography and particle size analysis.

Table 10
Amylose content in transgenic wheat lines determined by iodometric method Line Target enzyme Transformant No. Amylose Content (%) HB1 Untransformed - 31.8 144008 SBEIIb IIb 110.16b 27.3 144087 SBEIIa IIa 85.3a 88.5 144025 SBEIIa IIa 50.1b 75.8 Lsd - - 7.7

Example 15. Analysis for the distribution of chain lengths

The distribution of chain lengths of starch samples was determined by carbohydrate electrophoresis with fluorophore (FACE) after starch isoamylase was branched. The percentage of chain lengths of DP 6-11, DP 12-30 and DP 31-60 in starch of transgenic seeds was compared with the percentage of chain lengths of non-transgenic control. The results are shown in table 11. In Fig.21 presents graphs of molar differences, where the normalized distribution of chain lengths for starch of transgenic lines with a high content of amylose was subtracted from the normalized distribution of chain lengths for starch of isogenic non-transformed control.

Table 11
Distribution of chain lengths of isoamylase-branched starch derived from transgenic wheat lines Line Target gene Transformant No. DP4-12 DP13-24 DP24-36 > 36 HB1 Untransformed control - 57.39 37.38 3.83 1.40 144087 SBEIIa IIa 85.3a 47.40 42.27 6.16 4.17 144025 SBEIIa IIa 50.1b 49.99 44.40 5.60 - 144008 SBEIIb IIb 110.16b 57.98 37.65 4.37 -

There was a significant decrease in the ratio of the number of chains with a length of DP 4-12 in starch of ds-SBEIIa transgenic seeds compared to the number of chains with such a length in starch from non-transformed seeds or ds-SBEIIb transgenic seeds. The number of chains with a length> DP 13 was higher in ds-SBEIIa-transgenic seeds compared to other seeds. These results suggest that SBEIIa may be selectively involved in the synthesis of shorter DP4-12 chains in wheat starch. However, an increase in the content of shorter amylose chains was observed in the starch of the SSIIa mutant.

Example 16. The properties of starch transgenic wheat

The physical properties of starch of ds-SBEIIa- and ds-SBEIIb-transgenic lines, including gelatinization temperature, were analyzed by differential scanning calorimetry using a Perkin-Elmer Diamond calorimeter. About 20 mg of each starch was mixed with water in a ratio of 1: 2, that is, until the moisture content reached 66.7%, and hermetically closed in a DSC cell. The heating rate of the test samples was 10 ° C per minute, and the control samples from 0 to 150 ° C. The data obtained were analyzed using available software.

In the DSC thermogram for each starch, two peaks of the endotherm were observed. The first peak was the destruction of the crystalline structure during the gelatinization of starch. The second peak represented the endotherm of amylose-lipid dissociation. The maximum gelatinization temperature of starch obtained from dc-SBEIIa transgenic lines was approximately 7-10 ° C higher than the maximum starch temperature of the non-transformed control and approximately 3-7 ° C higher than the starch temperature of dc-SBEIIb transgenic line .

Table 12
Thermal properties of transgenic wheat starch, determined on a differential scanning calorimeter (DSC) Lines Target enzyme Peak 1 (gelatinization) Peak 2 (amylose-lipid dissociation) Start Peak the end Area □ N Start Peak the end □ N 008 SBEIIb 58.8 63.7 70.8 234.8 4,5 93.2 103.5 110.3 0.7 012 SBEIIb 59.0 64.1 70.8 262.6 4.3 94.5 103.1 109.7 0.6 121 SBEIIa 53.7 67.5 86.9 156.4 2.6 92.4 102.9 108.9 0.7 087 SBEIIa 53.1 71.9 85.9 142.6 2,4 95.7 102.7 108.9 0.7 114 SBEIIa 53.0 68.1 88.0 125.2 2.1 92.8 102.5 109.6 0.8 109c The control 55.9 60.7 68.8 234.3 3.9 97.2 104.6 109.9 0.4

In these lines, there was a noticeable increase in the final gelatinization temperature (first peak), approximately 16-19 ° C, compared with the gelatinization temperature of the untransformed control and ds-SBEIIb-transgenic lines. Obviously, the gelation onset temperature was lower for ds-SBEIIa transgenic lines compared to the control temperature or ds-SBEIIb transgenic lines. Ng et al. (1997) reported that the gelatinization onset temperature of corn starch having extended chain amylose (ae) was similar to the gelatinization onset temperature of normal corn starch, but there was a significant increase in the maximum gelatinization temperature in ae starch compared to normal starch. The gelatinization enthalpy of starch from ds-SBEIIa transgenic lines was significantly lower than the gelatinization enthalpy of control starch and starch from ds-SBEIIb lines. This, obviously, is reflected in a significant decrease in the gelatinization peak area, which is a reduced amount of amylopectin in dc-SBEIIa transgenic lines. In all transgenic lines, no significant change in the peak of amylose-lipid dissociation was observed. Therefore, the starch obtained by the authors had a number of these new properties.

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Claims (64)

1. A grain obtained from a wheat plant containing starch having a genetic modification, which leads to a decrease in the expression level of the SBEIIa gene, the activity of the SBEIIa enzyme in the endosperm, or both compared to wild-type grain, where this genetic modification includes a mutation of the SBEIIa gene or an embedded nucleic acid encoding an SBEIIa gene expression inhibitor, wherein the amylose level of the starch of said grain is at least 50%. 2. The grain obtained from a wheat plant containing starch in the grain and genetic modification, which leads to a decrease in the expression level of the SBEIIa gene, the activity of the SBEIIa enzyme in the endosperm, or both compared to wild-type grain, where this genetic modification includes a mutation of the SBEIIa gene or an integrated nucleic acid encoding an SBEIIa gene expression inhibitor, and wherein the amylose content of the starch of said grain is at least 30%. 3. The grain according to claim 2, where the specified genetic modification includes a mutation in the SBEIIa gene, leading to a decrease in the level of SBEIIa protein, the activity of the SBEIIa enzyme in the endosperm, or both compared to wild-type grain. 4. A grain according to any one of claims 1 to 3, containing a null mutation in at least one SBEIIa gene. 5. The grain according to claim 4, where the specified plant is hexaploid and where the specified grain contains zero mutations in two or three SBEIIa genes. 6. The grain according to claim 1 or 2, containing a transgene encoding an inhibitor of expression of the SBEIIa gene. 7. The grain according to claim 6, where the specified transgene encodes an antisense, cosuppressive, ribozyme or duplex RNA molecule. 8. The grain according to claim 1, which, in addition, contains a reduced level of SBEIIb protein, enzyme activity, or has both of these properties compared to wild-type grain. 9. The grain according to claim 1, containing a reduced level of SBEIIa protein, enzyme activity, or has both of these properties compared to wild-type grain. 10. The grain according to claim 1, containing a changed level of the enzyme compared to wild-type grain, where the specified enzyme is selected from the group consisting of ADP-glucose pyrophosphorylase, GBSS, SSI, SSII, SSIII, a branching enzyme such as isoamylase, a branching enzyme such as pullulanase and any combination of them. 11. The grain according to claim 2 or 3, where the amylose content in the starch of the grain is at least 40%. 12. The grain according to claim 1, which is non-wrinkled, or whose average weight is at least about 36 mg, or which has both properties. 13. The grain according to claim 1, where at least 50% of the starch granules obtained from the indicated grain are not birefringent when observed in polarized light. 14. The grain according to claim 1, where the starch content in the refined grain is at least 25% (wt./wt.) Or where the starch content is at least 90% or both compared with the content starch in wild-type grain. 15. The grain according to claim 1, where the specified grain is a whole grain or ground, crushed, crumbled, rolled, threshed, singed or broken grain. 16. A wheat plant capable of producing grain according to any one of claims 1 to 15. 17. Starch granules extracted from grain according to any one of claims 1 to 15. 18. The starch granules according to claim 17, comprising starch, wherein the amylose level in the starch of said granules is at least 50%. 19. Starch extracted from grain according to any one of claims 1 to 15. 20. A product containing grain according to any one of claims 1 to 15 or flour, whole grain flour, cereals or starch obtained from grain according to any one of claims 1 to 15, or any combination thereof. 21. The product according to claim 20, where the specified flour, whole grain flour, grits or starch are mixed with flour, whole grain flour, grits or starch obtained from another source. 22. The product according to claim 20, which is a non-food product. 23. A composition comprising starch according to claim 19 and another food ingredient or water. 24. The method of producing grain according to any one of claims 1 to 15, where the specified method includes the steps of:
i) introducing a genetic modification into a parent wheat or seed plant, wherein said genetic modification comprises a mutation of the SBEIIa gene or an integrated nucleic acid encoding an inhibitor of expression of the SBEIIa gene, and
ii) identification of a hereditary plant, or seeds of a parent wheat plant, or a seed that has a reduced level of expression of the SBEIIa gene, activity of the SBEIIa enzyme in the endosperm, or both compared to wild-type grain,
where the specified grain contains starch and where the amylose content in the starch of the grain is at least 50%. 25. The method of claim 24, wherein the step of introducing the genetic modification comprises administering an exogenous nucleic acid that expresses an inhibitor of expression of the SBEIIa gene. 26. The method according to paragraph 24, where the stage of introducing the genetic modification provides for the mutagenesis of the parent wheat or seed plant under the influence of a chemical agent or radiation. 27. The method of producing grain according to any one of claims 1 to 15, where the specified method includes the steps of:
i) introducing a genetic modification into a parent wheat plant or seed, wherein said genetic modification contains a mutation of the SBEIIa gene,
ii) identification of a hereditary plant, or a seed of a parent wheat plant, or a seed that has a reduced level of expression of the SBEIIa gene, activity of the SBEIIa enzyme in the endosperm, or both compared to wild-type grain,
iii) introducing a genetic modification into a parent wheat plant or seed, wherein said genetic modification contains a mutation of the SBEIIb gene,
iv) identifying a progeny plant, or a seed of a parent wheat plant, or a seed that has a reduced level of expression of the SBEIIb gene, activity of the SBEIIb enzyme in the endosperm, or both compared to wild-type grain;
v) crossing a plant having a reduced level of expression of the SBEIIa gene, activity of the SBEIIa enzyme in the endosperm or both, with a plant having a reduced level of expression of the SBEIIb gene, activity of the SBEIIb enzyme in the endosperm or both; and
identify wheat plants with a reduced level of expression of SBEIIa and SBEIIb genes, the activity of these enzymes, or both. 28. A method of obtaining a modified wheat starch, comprising the step of extracting starch from grain according to any one of claims 1 to 15. 29. The wheat plant according to clause 16, which is a Triticum aestivum ssp.aestivum. 30. A grain obtained from a wheat plant containing a mutation where the SBEIIa gene is absent in the long arm of chromosome 2A or where the SBEIIa gene present on the long arm of chromosome 2A contains a mutation leading to a decrease in SBEIIa protein, SBEIIa enzyme activity, or both in the endosperm of the indicated grain compared to wild-type grain. 31. The grain of claim 30, wherein said mutation is a null mutation of the SBEIIa gene. 32. The grain of claim 30 or 31, wherein said mutation is a deletion of at least a portion of the SBEIIa gene. 33. The grain of claim 30, which further comprises a mutation where the SBEIIb gene is absent on the long arm of chromosome 2A or where the SBEIIb gene present on the long arm of chromosome 2A contains a mutation that leads to a decrease in SBEIIb protein, SBEIIb enzyme activity, or both different in the endosperm of said grain compared to wild-type grain. 34. The grain of claim 30, wherein said deletion inhibits the expression of SBEIIa and SBEIIb genes on the long arm of chromosome 2A. 35. The grain of claim 30, wherein said plant is a durum wheat plant. 36. The grain of claim 35, further comprising a genetic modification leading to a decrease in the activity of the starch-branching enzyme encoded by the SBEIIa gene on the long arm of chromosome 2B, compared to wild-type grain. 37. The grain according to clause 36, where the indicated genetic modification is the absence of the SBEIIa gene on the long arm of chromosome 2B or a mutation of the SBEIIa gene on the long arm of chromosome 2B, which leads to a decrease in the activity of the SBREIIa enzyme in the endosperm of the indicated grain compared to wild-type grain. 38. The grain of claim 30, wherein said plant is the plant Triticum aestivum ssp.aestivum. 39. The grain of claim 38, comprising a genetic modification leading to a decrease in the activity of the starch-branching enzyme encoded by the SBEIIa gene on the long arm (s) of chromosome 2B, chromosome 2D, or both chromosomes, compared to wild-type grain. 40. The grain according to § 39, where the specified genetic modification is the absence of the SBEIIa gene in at least one of these chromosomes or a mutation of the SBEIIa gene in at least one of these chromosomes, which leads to a decrease in the activity of the SBEIIa enzyme in the endosperm specified grain in comparison with wild-type grain. 41. The grain of claim 36, wherein said genetic modification comprises introducing a nucleic acid encoding an SBEIIa gene expression inhibitor, SBEIIa enzyme activity, or both. 42. The grain according to item 30, where the level of activity of the enzyme SBEIIa is at least 40% lower compared to wild-type grain. 43. The grain of claim 30, wherein the amylose content of the starch of said grain is at least 30%. 44. The grain according to item 43, where the amylose content in the starch of the specified grain is at least 50%. 45. The grain according to claim 1, where the amylopectin of the specified grain has a reduced content of dp fractions with a chain length of 4-12 units compared to amylopectin of wild-type grain according to the measurement results after amylopectin isoamylase is branched. 46. The grain of claim 30, which is non-wrinkled. 47. The grain of claim 30, which has an average weight of at least about 36 mg. 48. The grain according to item 43, where at least 50% of the starch granules from the specified grain are non-birefringent when observed in polarized light. 49. The grain according to paragraph 41, where the starch content in the refined grain is at least 25% (wt./wt.) Or at least 90% of the starch content in wild-type grain. 50. The grain of claim 30, wherein said grain is whole grain or ground, crushed, crushed, crushed, rolled, threshed, scorched or beaten grain. 51. The grain according to claim 30, which, in addition, contains a reduced level of SBEI protein, activity of the SBEI enzyme, or both in comparison with wild-type grain. 52. The grain of claim 31, further comprising a changed level of the enzyme compared to the level of the wild-type grain enzyme, wherein said enzyme is selected from the group consisting of ADP-glucose pyrophosphorylase, GBSS, SSI, SSII, SSIII, a branching isoamylase-type enzyme, a branching enzyme type pullulanase and any combination thereof. 53. A wheat plant capable of producing grain according to any one of paragraphs.30-52. 54. Starch granules extracted from wheat grain according to any one of paragraphs.30-52. 55. The starch granules according to item 54, containing starch, in which the level of amylose in the starch of the granules is at least 50%. 56. Starch extracted from grain according to any one of paragraphs.30-52. 57. A product containing grain according to any one of paragraphs.30-52 or flour, whole grain flour, cereals or starch obtained from grain according to any one of paragraphs.30-52. 58. The product according to § 57, where the flour, whole grain flour, grits or starch are mixed with flour, whole grain flour, grits or starch obtained from another source. 59. The product according to § 57, which is a non-food product. 60. A composition containing starch according to item 56 and another food ingredient or water. 61. A method of producing grain, comprising (i) growing a wheat plant and (ii) collecting grain from said wheat plant, where said grain contains a mutation, where the SBEIIa gene is absent on the long arm of chromosome 2A or where the SBEIIa gene is present on the long arm of chromosome 2A contains a mutation leading to a decrease in the activity of the enzyme SBEIIa in the endosperm of the indicated grain compared to wild-type grain. 62. A method for producing starch, comprising (i) obtaining grain according to any one of claims 30-52; and (ii) extracting starch from said grain.
Priority on points: 07/01/2003 according to claims 1-29; 06/30/2004 according to claims 30-62.

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